Use of rgm and its modulators

ABSTRACT

The present invention relates to the use of a modulator of a polypeptide having or comprising an amino acid sequence as disclosed herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with seizures or associated with angiogenic disorders or disorders of the cardio-vascular system. Furthermore, the invention provides for the use of a modulator of a polypeptide having or comprising said amino acid sequence of of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with angiogenic disorders or disorders of the cardio-vascular system. In addition the invention provides for the use of said polypeptide or said functional fragment or derivative thereof for the preparation of a pharmaceutical composition for preventing or treating tumor growth or formation of tumor metastases or as a marker of stem cells.

[0001] The present invention relates to the use of a modulator of apolypeptide having or comprising an amino acid sequence as disclosedherein or of a functional fragment or derivative thereof or of apolynucleotide encoding said polypeptide or fragment or derivative forthe preparation of a pharmaceutical composition for preventing,alleviating or treating diseases or conditions associated with thedegeneration or injury of vertebrate nervous tissue, associated withangiogenic disorders or disorders of the cardio-vascular system.Furthermore, the invention provides for the use of a modulator of apolypeptide having or comprising said amino add sequence or of afunctional fragment or derivative thereof or of a polynucleotideencoding said polypeptide or fragment or derivative for the preparationof a pharmaceutical composition for preventing, alleviating or treatingdiseases or conditions associated with the degeneration or injury ofvertebrate nervous tissue, associated with seizures, associated withangiogenic disorders or disorders of the cardiovascular system. Inaddition the invention provides for the use of said polypeptide or saidfunctional fragment or derivative thereof for the preparation of apharmaceutical composition for preventing or treating tumor growth orformation of tumor metastases or as a marker of stem cells.

[0002] Several documents are cited throughout the text of thisspecification. The disclosure content of each of the documents citedherein (including any manufacturer's specifications, instructions, etc.)are hereby incorporated by reference.

[0003] The most important mechanism in formation of embryonic nervoussystems is the guidance of axons and growth cones by directionalguidance cues (Goodman, Annu. Rev. Neurosci. 19 (1996), 341-77; Mueller,Annu. Rev. Neurosci 22, (1999), 351-88). A suitable model system forstudying this guidance process is the retinotectal system ofvertebrates. In the chick embryo approximately 2 million retinalganglion cell (RGC) axons leave each eye and grow towards thecontralateral tectum opticum to form a precise map (Mey & Thanos,(1992); J. Himforschung 33, 673-702). Having arrived at the anteriorpole of the optic tectum, RGC axons start to invade their tectal targetto find their target neurons. Mapping occurs in such a way, that RGCaxons from nasal retina project to posterior tectum and temporal axonsto anterior tectum. Along the dorso-ventral axis, axons coming fromdorsal retina terminate in ventral tectum, whereas those from ventralretina end up in dorsal tectum. In the end a precise topographic map isformed, where neighborhood relationships in the retina are preserved inthe tectum, so that axons from neighboring ganglion cells in the retinasynapse with neighboring tectal neurons. Most important for formation ofthis map, are graded tectal guidance cues, read by retinal growth conescarrying corresponding receptors which also show a graded distribution(Sperry, Proc. Natl. Acad. Sci. USA 50 (1963), 703-710; Bonhoeffer &Gierer, Trends Neurosci. 7 (1984) 378-381). Position of each retinalgrowth cone in the tectal field is therefore determined by two sets ofgradients: receptor gradients on ingrowing retinal axons and growthcones and ligand gradients on tectal cells (Gierer, Development 101(1987), 479-489). The existence of the graded tectal ligands has beenpostulated from anatomical work, their identification however proved tobe extremely difficult and was only made possible with the developmentof simple in vitro systems (Walter, Development 101 (1987), 685-96; Cox,Neuron 4 (1990), 31-7). In the stripe assay RGC axons grow on a membranecarpet, consisting of alternating lanes of anterior (a) and posterior(p) tectal membranes. On these carpets, temporal retinal axons grow onanterior tectal membranes and are repelled by the posterior lanes,whereas nasal axons do not distinguish between a and p membranes(Walter, Development 101 (1987), 685-96). The same specificity is alsoobserved in the growth cone collapse assay (Raper & Kapfhammer, Neuron 4(1990), 21-29), where temporal retinal growth cones collapse afteraddition of posterior tectal membrane vesicles but do not react toanterior tectal vesicles and where nasal growth cones are insensitive toeither type of vesicles (Cox, (1990), loc. cit.). In both assay systems,treatment of posterior tectal membranes with the enzymephosphatidylinositol-specific phospholipase C (PI-PLC) which cleaves thelipid anchor of glycosylphosphatidylinositol (GPI)-linked proteins,removed their repellent and collapse-inducing activity (Walter, J.Physiol 84 (1990), 104-10).

[0004] One of the first repulsive guidance molecules identified in theretinotectal system of chick embryos was a GPI-anchored glycoproteinwith a molecular weight of 33/35 kDa (Stahl, Neuron 5 (1990), 735-43).This 33/35 kDa molecule, later termed RGM (Repulsive Guidance Molecule),was active in both stripe and collapse-assays and was shown to beexpressed in a low-anterior high-posterior gradient in the embryonictecta of chick and rat (Mueller, Curr. Biol. 6 (1996), 1497-502;Mueller, Japan Scientific Societies Press (1997), 215-229). Due to theabnormal biochemical behaviour of RGM, the precise amino acid sequencewas not obtainable. RGM was described as a molecule which is activeduring vertebrate development. Interestingly, RGM is down-regulated inthe embryonic chick tectum after E12 and in the embryonic rat tectumafter P2 and completely disappears after the embryonic stages (Müller(1992), Ph. D thesis University of Tübingen; Müller (1997) JapanScientific Societies, 215-229) In 1996, Müller (loc. cit) have shownthat CALI (chromophore-assisted laser inactivation) of RGM eliminatesthe repulsive guidance activity of posterior tectal membranes /RGM.However, due to the presence of other guidance molecules, in particularof RAGS (repulsive axon guidance signal) and ELF-1 (Eph ligand family1), a complete elimination of guidance was not always detected and itwas speculated that RGM acts in concert with RAGS (now termed ephrin-A5)and ELF-1 (ephrin-A2). It was furthermore envisaged that RGM may be aco-factor potentiating the activity of RAGS and ELF-1 in embryonicguidance events.

[0005] In 1980/81 the group of Aguayo found that, when peripheralneurons are transplanted/grafted into injured CNS of adult, axon growthof CNS neurons is induced (David, Science 214 (1981), 931-933).Therefore, it was speculated that CNS neurons have still the ability andcapacity of neurite-outgrowth and/or regeneration, if a suitableenvironment would be provided. Furthermore, it was speculated that“CNS-neuron regeneration inhibitors” may exist.

[0006] In 1988, Caroni and Schwab (Neuron 1, 85-96) described twoinhibitiors of 35 kDa and 250 kDa, isolated from rat CNS myelin (NI-35and NI-250; see also Schnell, Nature 343 (1990) 269-272; Caroni, J. CellBiol. 106 (1988), 1291-1288).

[0007] In 2000, the DNA encoding for NI-220/250 was deduced and thecorresponding potent inhibitor of neurite growth was termed Nogo-A(Chen, Nature 403 (2000), 434-438. The membrane-bound Nogo turned out tobe a member of the reticulon family (GrandPré, Nature 403 (2000),439-444).

[0008] Further factors which mediate neuronal outgrowth inhibition havefirst been isolated in grasshoppers, and termed ,,fasciclin IV” andlater ,,collapsin” in chicken. These inhibitors belong to the so-calledsemaphorin family. Semaphorins have been reported in a wide range ofspecies and described as transmembrane proteins (see, inter alia,Kolodkin Cell 75 (1993) 1389-99, Püschel, Neuron 14 (1995), 941-948).Yet, it was also shown that not all semaphorins have inhibitoryactivity. Some members of said family, e.g. semaphorin E, act as anattractive guidance signal for cortical axons (Bagnard, Development 125(1998), 5043-5053).

[0009] A further system of repulsive guidance molecules is theephrin-Eph system. Ephrins are ligands of the Eph receptor kinases andare implicated as positional labels that may guide the development ofneural topographic maps (Flanagan, Ann. Rev. Neurosc. 21 (1998),309-345). Ephrins are grouped in two classes, the A-ephrins which arelinked to the membrane by a glycosylphosphatidylinositol-anchor(GPI-anchor) and the B-ephrins carrying a transmembrane domain (Ephnomenclature committee 1997). Two members of the A-ephrins, ephrin-A2and ephrin-A5, expressed in low anterior-high posterior gradients in theoptic tectum, have recently been shown to be involved in repulsiveguidance of retinal ganglion cell axons in vitro and in vivo (see, interalia (Drescher, Cell 82 (1995), 359-70; Cheng, Cell 79 (1994), 157-168;Feldheim, Neuron 21 (1998), 563-74; Feldheim, Neuron 25 (2000), 563-74)

[0010] Considering the fact that a plurality of physiological disordersor injuries are related to altered cellular migration processes, thetechnical problems underlying the present invention was to provide formeans and methods for modifying altered developmental or cellular(migration) processes which lead to disease conditions.

[0011] Accordingly, the present invention relates to the use of anmodulator of a polypeptide having or comprising the amino acid sequenceof SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment or derivativethereof or of a polynucleotide encoding said polypeptide or fragment orderivative for the preparation of a pharmaceutical composition forpreventing, alleviating or treating diseases or conditions associatedwith the degeneration or injury of vertebrate nervous tissue, associatedwith angiogenic disorders or disorders of the cardiovascular system andassociated with tumor formation and tumor growth.

[0012] In context of the present invention, and as documented in theappended examples, it was surprisingly found that the repulsive guidancemolecule (RGM) is not only expressed during vertebrate development butis re-expressed in adult tissue, in particular in damaged adult tissues.It was, inter alia, surprisingly found that RGM is re-expressed afterdamage of the nervous tissue, after traumatic events or focal ischemias.The present invention provides for the complete nucleotide sequenceand/or amino acid of RGM (see, e.g. SEQ ID NO: 17 or 18 depicting theRGM sequence of chicken or SEQ ID NO: 20 to 25 depicting the human RGMhomologues.) RGM, as pointed out herein above, is a glycoprotein, linkedto membranes by a GPI-anchor.

[0013] Said GPI-anchor also carries a cross-reacting determinant (CRD)epitope and its carbohydrate part is able to bind peanut lectin. Asdocumented herein, the RGM protein is a potent growth inhibitor and canassert neurite growth inhibition in picomolor concentrations.

[0014] The term “modulator” as employed herein relates to “inhibitors”as well as “activators” of RGM function. Most preferably said“modulation” is an inhibition, wherein said inhibition may be a partialor a complete inhibition.

[0015] The term ,,amino acid sequence of SEQ ID NO: 18, 20, 23 or 25 asemployed herein relates to the amino acid sequence of RGM (repulsiveguidance molecule) and relates to the RGM polypeptide of chicken orhuman, respectively. In particular, SEQ ID NOs: 20 and 21 depict humanRGM1. Human RGM1 has been localized on chromosome 15. Further, humanRGMs comprise RGM2 and RGM3. RGM2 is depicted in SEQ ID NO: 23 (aminoacid sequence) and is encoded by a nucleotide sequence as shown in SEQID NO: 22. Human RGM2 has been localized on chromosome 5. Furthermore,human RGM3 is shown in appended SEQ ID NO: 25 (amino acid sequence) andencoded by a nucleotide sequence as depicted in SEQ ID NO: 24. HumanRGM3 is located on chromosome 1. Yet, as will be discussed herein below,said term relates also to further RGM homologues.

[0016] The term “(poly)peptide” means, in accordance with the presentinvention, a peptide, a protein, or a (poly)peptide which encompassesamino acid chains of a given length, wherein the amino acid residues arelinked by covalent peptide bonds. However, peptidomimetics of such RGMproteins/(poly)peptides wherein amino acid(s) and/or peptide bond(s)have been replaced by functional analogs are also encompassed by theinvention.

[0017] The present invention is not restricted to RGM from human, mouseor chicken and its inhibitors but also relates to the use of inhibitorsof RGM or of RGM itself (or functional fragments or derivatives thereof)from other species. Since the present invention provides for the use ofamino acid seuqences/polypeptides of RGM and its correspondinginhibitors and since the amino acid sequences of human and chicken RGMare disclosed herein, the person skilled in the art is provided with theinformation to obtain RGM sequences from other species, like, interalia, mouse, rat, pig, etc. The relevant methods are known in the artand may be carried out by standard methods, employing, inter alia,degenerate and non degenerate primers in PCR-techniques. Such molecularbiology methods are well known in the art and, e.g., described inSambrook (Molecular Cloning; A Laboratory Manual, 2^(nd) Edition, ColdSpring Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989)) andAusubel, “Current Protocols in Molecular Biology”, Green PublishingAssociates; and Wiley Interscience, N.Y. (1989).

[0018] Furthermore, as employed in the context of the present invention,the term “RGM”, “RGM modulator” and “RGM-inhibitor” also relates to RGMmolecules (and their corresponding inhibitors) which are variants orhomologs of the RGM molecules (and their inhibitors) as describedherein. “Homology” in this context is understood to refer in thiscontext to a sequence identity of RGMs of at least 70%, preferably morethan 80% and still more preferably more than 90% on the amino acidlevel. The present invention, however, comprises also (poly)peptidesdeviating from wildtype amino acid sequences of human or chicken RGMsdescribed herein, wherein said deviation may be, for example, the resultof amino acid and/or nucleotide substitution(s), deletion(s),addition(s), insertion(s), duplication(s), inversion(s) and/orrecombination(s) either alone or in combination. Those deviations maynaturally occur or be produced via recombinant DNA techniques well knownin the art. The term “variation” as employed herein also comprises“allelic variants”. These allelic variations may be naturally occurringallelic variants, splice variants as well as synthetically produced orgenetically engineered variants.

[0019] The term “polynucleotide” in accordance with the presentinvention comprises coding and, wherever applicable, non-codingsequences (like promotors, enhancers etc.). It comprises DNA, RNA aswell as PNA. In accordance with the present invention, the term“polynucleotide/nucleic acid molecule” comprises also any feasiblederivative of a nucleic acid to which a nucleic acid probe mayhybridize. Said nucleic acid probe itself may be a derivative of anucleic acid molecule capable of hybridizing to said nucleic acidmolecule or said derivative thereof. The term “nucleic acid molecule”further comprises peptide nucleic acids (PNAs) containing DNA analogswith amide backbone linkages (Nielsen, Science 254 (1991), 1497-1500).The term “nucleic acid molecule” which encodes a RGM (poly)peptide or afunctional fragment/derivative thereof, in connection with the presentinvention, is defined either by (a) the specific nucleic acid sequencesencoding said (poly)peptide specified in the present invention or (b) bynucleic acid sequences hybridizing under stringent conditions to thecomplementary strand of the nucleotide sequences of (a) and encoding a(poly)peptide deviating from the nucleic acid of (a) by one or morenucleotide substitutions, deletions, additions or inversions and whereinthe nucleotide sequence shows at least 70%, more preferably at least 80%identity with the nucleotide sequence of said encoded RGM (poly)peptidehaving an amino acid sequence as defined herein above and functions as aRGM (or a functional fragment/derivative thereof).

[0020] The term “modulator” as employed herein also comprises the term“inhibitor”, as mentioned herein above.

[0021] The term “inhibitor of a polypeptide having or comprising theamino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or a functionalfragment or derivative thereof”, therefore, not only relates to thespecific inhibitors of human or chicken RGM but also relates toinhibitors of RGM (or functional fragments or derivatives thereof ofother species. Useful inhibitors are disclosed herein as well asdescribed herein below and in the appended examples.

[0022] The term “inhibitor” also comprises “modulators” of the RGMpolypeptides and/or the RGM encoding nucleic acid molecule/gene. Incontext of this invention it is also envisaged that said “modulation”leads, when desired, to an activation of RGM.

[0023] The term “functional fragment or derivative thereof” in contextof the present invention and in relation to the herein described RGMmolecules comprises fragments of the RGM molecules defined herein havinga length of at least 25, more preferably at leat 50, more preferably atleast 75, even more preferably at least 100 amino acids. Functionalfragments of the herein identified RGM molecules or RGM molecules ofother species (homologous RGMs) may be comprised in fusion and/orchimeric proteins. “Functional fragments” comprise RGM fragments (or itsencoding nucleic acid molecules) which are able to replace RGM fulllength molecules in corresponding assays (as disclosed herein in theappended examples, e.g. collapse and/or stripe assays) or may elucidatean anti-RGM specific immune-response and/or lead to specific anti-RGMantibodies. An example of such a “functional fragment” is, inter alia,the functional fragment of chicken RGM depicted in SEQ ID NO: 19. Incontext of the present invention, polynucleotides encoding functionalfragments of RGM and/or its derivatives have preferably at least 15,more preferably at least 30, more preferably at least 90, morepreferably of at least 150, more preferably of at least 300 nucleotides.The term “derivative” means in context of their invention derivatives ofRGM molecules and/or their encoding nucleic acid molecules and refer tonatural derivatives (like allelic variants) as well as recombinantlyproduced derivatives/variants which may differ from the herein describedRGM molecules by at least one modification/mutation, e.g. at least onedeletion, substitution, addition, inversion or duplication. The term“derivative” also comprises chemical modifications. The term“derivative” as employed herein in context of the RGM molecule alsocomprises soluble RGM molecules which do not comprise any membraneanchorage.

[0024] As mentioned herein above, the present invention provides for theuse of a modulator, preferably an inhibitor, of RGM molecules and/ortheir corresponding encoding polynucleotides/nucleic acid molecules forthe preparation of a pharmaceutical composition for preventing,alleviating or treating various disorders of the nervous system,angiogenic disorders or disorders of the cardiovascular system andmalignancies of different etiology.

[0025] In a preferred embodiment, said disorders of the nervous systemcomprise degeneration or injury of vertebrate nervous tissue, inparticular neurodegenerative diseases, nerve fiber injuries anddisorders related to nerve fiber losses.

[0026] Said neurodegenerative diseases may be selected from the groupconsisting of motorneuronal diseases (MND), amyotrophic lateralsclerosis (ALS), Alzheimers disease, Parkinsons disease, progressivebulbar palsy, progressive muscular atrophy, HIV-related dementia andspinal muscular atrophy(ies), Down's Syndrome, Huntington's Disease,Creutzfeldt-Jacob Disease, Gerstmann-Straeussler Syndrome, kuru,Scrapie, transmissible mink encephalopathy, other unknown priondiseases, multiple system atrophy, Riley-Day familial dysautonomia saidnerve fiber injuries may be selected from the group consisting of spinalcord injury(ies), brain injuries related to raised intracranialpressure, trauma, secondary damage due to increased intracranialpressure, infection, infarction, exposure to toxic agents, malignancyand paraneoplastic syndromes and wherein said disorders related to nervefiber losses may be selected from the group consisting of paresis ofnervus facialis, nervus medianus, nervus ulnaris, nervus axillaris,nervus thoracicus longus, nervus radialis and for of other peripheralnerves, and other aquired and non-aquired deseases of the (human)central and peripheral nervous system.

[0027] The above mentioned spinal cord and brain injuries not onlycomprise traumatic injuries but also relate to injuries caused bystroke, ischemia and the like. It is in particular envisaged that theinhibitors as defined herein below and comprising, inter alia, anti-RGMantibodies be employed in the medical art to stimulate nerve fibergrowth in individuals, in particular in vertebrates, most preferably inhumans.

[0028] In a more preferred embodiment of the present invention, theinvention provides for the use of a modulator, preferably an inhibitorto RGM (or a functional fragment or derivative thereof) for thepreparation of a pharmaceutical composition for the treatment ofdisorders of the cardiovascular system, wherein these disorders, e.g.,comprise disorders of the blood-brain barrier, brain oedema, secondarybrain damages due to increased intracranial pressure, infection,infarction, ischemia, hypoxia, hypoglycemia, exposure to toxic agents,malignancy, paraneoplastic syndromes.

[0029] It is envisaged, without being bound by theory, that RGMinhibitors may stimulate surviving neurons to project collateral fibersinto the diseased tissue, e.g. the ischemic tissue.

[0030] As illustrated in the appended examples, RGM is expressed locallyat the side of artificial transection of brain/spinal cord tissue intest animals (like rats), e.g., in the penumbra region surrounding anischemic core of a human suffering focal ischemia in the temporalcontex. Furthermore, it is documented in the appended examples that RGMis, surprisingly, expressed in tissue(s) having experienced fromtraumatic brain injuries. The invention also relates to the use of a RGMpolypeptide or a functional fragment or derivative thereof or the use ofa polynucleotide encoding the same (polypeptides and polynucleotides asdefined herein), wherein the above described disease or conditionassociated with seizures is epilepsy. An epilepsy is therebycharacterized by an epileptic seizure as a convulsion or transientabnormal event experienced by the subject, e.g. a human patient, due toa paroxysmal discharge of (cerebral) neurons. The epileptic seizurescomprise tonic seizures, tonic-clonic seizures (grand mal), myoclonicseizures, absence seizures as well as akinetic seizures. Yet, alsocomprised are in context of this invention simple partial seizures, e.g.Jacksonian seizures and seizures due to perinatal trauma and/or fetalanoxia. As mentioned herein below, the uses described herein relate inparticular to the preparation of pharmaceutical compositions for thetreatment of diseases/conditions associated with aberrant sprouting ofnerve fibres, like epilepsy; see also Routbort, Neuroscience 94 (1999),755-765.

[0031] In a even more preferred embodiment of the invention, themodulator, preferably the inhibitor of RGM (or of its functionalfragment or derivative thereof or of its encoding nucleid acid molecule)is used for the preparation of a pharmaceutical composition for themodification of neovascularization. Said modification may compriseactivation as well as stimulation. It is in particular envisaged thatsaid neovascularisation be stimulated and/or activated in diseasedtissue, like inter alia, ischemic and/or infarctious tissue.Furthermore, it is envisaged that the RGM-inhibitors described hereinmay be employed in the regulation of the blood-brain barrierpermeability.

[0032] It is furthermore envisaged that said modulators, preferably saidinhibitors for RGM be employed in the alleviation, prevention and/orinhibition of progression of vascular plaque formation (e.g.artherosclerosis) in cardiovascular, cerebo-vascular and/ornephrovascular diseases/disorders.

[0033] Furthermore, the present invention provides for the use of amodulator, preferably an inhibitor of RGM as defined herein for thepreparation of a pharmaceutical composition for remyelination.Therefore, the present invention provides for a pharmaceuticalcomposition for the treatment of demyelinating diseases of the CNS, likemultiple sclerosis or of demyelinating diseases like peripheralneuropathy caused by diphteria toxin, Landry-Guillain-Barré-Syndrom,Elsberg-Syndrom, Charcot-Marie-Tooth disease and other polyneuropatias.A particular preferred inhibitor of RGM in this context is an antibodydirected against RGM, e.g. an IgM antibody. It has previously be shownthat certain IgMs bind to oligodendrocytes and thereby induceremyelination. IgM antibodies against RGM are known in the art andcomprise e.g. the F3D4 described in the appended examples.

[0034] In addition the invention provides for the use of a RGMpolypeptide as defined herein or of a functional fragment or derivativethereof or of a polynucleotide encoding said polypeptide or fragment orderivative for the preparation of a pharmaceutical composition forpreventing, alleviating or treating diseases or conditions associatedwith the activity of autoreactive immune cells or with overactiveinflammatory cells. Most preferably these cells are T-cells.

[0035] Furthermore, the present invention relates to the use of amodulator, preferably an inhibitor or another RGM binding molecule of aRGM polypeptide or of a functional fragment or derivative thereof or ofa polynucleotide encoding said polypeptide or of fragment/derivativethereof for modifying and/or altering the differentiation status ofneuronal stem cells and/or their progenitors. Said stem cells arenormally found in the subventricular zones of many brain regions. It isknown that factors in the microenvironment of the brain dramaticallyinfluence the differentiation of undifferentiated stem cells. It isassumed that due to the characteristic expression of RGM in thesubventricular layers of many different brain regions, this moleculecould be a marker for stem cells. Furthermore, RGM inhibitors, likeantibodies could be useful markers for stem cells. Most important instem cell biology is the understanding of factors influencing theirdifferentiation. It is therefore assumed that RGM inhibitors change thedevelopmental fate of these cells.

[0036] As documented in the appended examples, RGM is not only expressedin ischemic tissue but is also expressed in scar tissue surrounding(brain) lesions.

[0037] It is particularly preferred that the modulator, preferably theinhibitior of the RGM molecule (or its functional fragment orderivative) is an antibody or a fragment or a derivative thereof, is anaptamer, is a specific receptor molecule capable of interacting with aRGM polypeptide or with a functional fragment or derivative thereof, oris a specific nucleic acid molecule interacting with a polynucleotideencoding an RGM and/or the polypeptide as defined herein.

[0038] The antibody to be used in context of the present invention canbe, for example, polyclonal or monoclonal antibodies. Techniques for theproduction of antibodies are well known in the art and described, e.g.in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. The production of specific anti-RGM antibodies isfurther known in the art (see, e.g. Müller (1996) loc.cit.) or describedin the appended examples.

[0039] The term “antibody” as employed herein also comprises chimeric,single chain and humanized antibodies, as well as antibody fragments,like, inter alia, Fab fragments.

[0040] Antibody fragments or derivatives further comprise F(ab′)₂, Fv orscFv fragments; see, for example, Harlow and Lane, loc.cit. Variousprocedures are known in the art and may be used for the production ofsuch antibodies and/or fragments, see also appended examples. Thus, the(antibody) derivatives can be produced by peptidomimetics. Further,techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to polypeptide(s) of this invention. Also, transgenicanimals may be used to express humanized antibodies to polypeptides ofthis invention. Most preferably, the antibody to be used in theinvention is a monoclonal antibody, for example the F3D4 antibodydescribed in the appended examples may be employed when an IgM isdesired. The general methodology for producing, monoclonal antibodies iswell-known and has been described in, for example, Köhler and Milstein,Nature 256 (1975), 494-496 and reviewed in J. G. R. Hurrel, ed.,“Monoclonal Hybridoma Antibodies: Techniques and Applications”, CRCPress Inc., Boco Raron, Fla. (1982), as well as that taught by L. T.Mimms et al., Virology 176 (1990), 604-619.

[0041] Preferably, said antibodies (or inhibitors) are directed againstfunctional fragments of the RGM polypeptide. As pointed out herein aboveand as documented in the appended examples, such functional fragmentsare easily deducible for the person skilled in the art and,correspondingly, relevant antibodies (or other inhibitors) may beproduced.

[0042] The “modulator”, preferably the “inhibitor” as defined herein mayalso be an aptamer.

[0043] In the context of the present invention, the term “aptamer”comprises nucleic acids such as RNA, ssDNA (ss=single stranded),modified RNA, modified ssDNA or PNAs which bind a plurality of targetsequences having a high specificity and affinity. Aptamers are wellknown in the art and, inter alia, described in Famulok, Curr. Op. Chem.Biol. 2 (1998), 320-327. The preparation of aptamers is well known inthe art and may involve, inter alia, the use of combinatorial RNAlibraries to identify binding sites (Gold, Ann. Rev. Biochem. 64 (1995),763-797). Said other receptors may, for example, be derived from saidantibody etc. by peptdomimetics.

[0044] Other specific “receptor” molecules which may function asinhibitors of the RGM polypeptides are also comprised in this invention.Said specific receptors may be deduced by methods known in the art andcomprise binding assays and/or interaction assays. These may, interalia, involve assays in the ELISA-format or FRET-format. Said“inhibitor” may also comprise specific peptides binding to and/orinterfering with RGM.

[0045] Furthermore, the above recited “modulator”, preferably“inhibitor” may function at the level of RGM gene expression. Therefore,the inhibitor may be a (specific) nucleic acid molecule interacting witha polynucleotide encoding a RGM molecule (or a functional fragment orderivative thereof.) These inhibitors may, e.g., comprise antisensenucleic acid molecules or ribozymes.

[0046] The nucleic acid molecule encoding RGM (and as disclosed herein,e.g., SEQ ID NO: 17) may be employed to construct appropriate anti-senseoligonucleotides. Said anti-sense oligonucleotides are able to inhibitthe function of wild-type (or mutant) RGM genes and comprise,preferably, at least 15 nucleotides, more preferably at least 20nucleotides, even more preferably 30 nucleotides and most preferably atleast 40 nucleotides.

[0047] In addition, ribozyme approaches are also envisaged for use inthis invention. Ribozymes may specifically cleave the nucleic acidmolecule encoding RGMs.

[0048] In the context of the present invention ribozymes comprise, interalia, hammerhead ribozymes, hammerhead ribozymes with altered coresequences or deoxyribozymes (see, e.g., Santoro, Proc. Natl. Acad. Sci.USA 94 (1997), 4262) and may comprise natural and in vitro selectedand/or synthesized ribozymes.

[0049] Nucleic acid molecules according to the present invention whichare complementary to nucleic acid molecules coding forproteins/(poly)peptides regulating, causing or contributing to obesityand/or encoding a mammalian (poly)peptide involved in the regulation ofbody weight (see herein below) may be used for the construction ofappropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201,EP-A2 0 360 257) which specifically cleave nucleic acid molecules of theinvention. Selection of the appropriate target sites and correspondingribozymes can be done as described for example in Steinecke, Ribozymes,Methods in Cell Biology 50, Galbraith, eds. Academic Press, Inc. (1995),449-460.

[0050] Said “inhibitor” may also comprise double-stranded RNAs, whichlead to RNA-mediated gene interference (see Sharp, Genes and Dev. 13(1999), 139-141)

[0051] Further potential inhibitors of RGM may be found and/or deducedby interaction assay and employing corresponding read-out systems. Theseare known in the art and comprise, inter alia, two hybrid screenings(as, described, inter alia, in EP-0 963 376, WO 98/25947, WO 00/02911)GST-pull-down columns, co-precipitation assays from cell extracts asdescribed, inter alia, in Kasus-Jacobi, Oncogene 19 (2000), 2052-2059,“interaction-trap” systems (as described, inter alia, in U.S. Pat. No.6,004,746) expression cloning (e.g. lambda gtII), phage display (asdescribed, inter alia, in U.S. Pat. No. 5,541,109), in vitro bindingassays and the like. Further interaction assay methods and correspondingread out systems are, inter alia, described in U.S. Pat. No. 5,525,490,WO 99/51741, WO 00/17221, WO 00/14271 or WO 00/05410.

[0052] A further objective of the present invention is to provide forthe use of a RGM polypeptide and/or of polypeptide having or comprisingthe amino acid sequence of SEQ ID NOs. 18, 20, 23 or 25 or of afunctional fragment or derivative thereof or of a polynucleotideencoding said polypeptide or fragment or derivative for the preparationof a pharmaceutical composition for preventing, alleviating or treatingdiseases or conditions associated with excessive collateral sprouting ofnerve fibres.

[0053] The present invention, therefore, provides for the medical use ofRGM protein(s) and/or functional fragments/derivatives thereof or forthe use of polynucleotides encoding said RGM protein(s) in conditionswhere excessive collateral sprouting occurs. Said conditions comprise,but are not limited to, epilepsy, phantom pain and neuropathic pain. Forexample, McNamara (Nat. Suppl. 399 (1999), A15-A22) has described thatsaid sprouting occurs in certain types of epilepsy. The RGM molecule,either naturally isolated or recombinantly produced, or its functionalfragments/derivatives may therefore be employed as potent “stop” signalsfor growing nerve fibres. The feasibility of such an approach has beenshown by Tanelian (Nat. Med. 3 (1997), 1398-1401) who employed asemaphorin for inhibition of nerve fiber growth.

[0054] In yet another embodiment, the present invention provides for theuse of RGM and/or of a polypeptide having or comprising the amino acidsequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment orderivative thereof or of a polynucleotide encoding said polypeptide orfragment or derivative for the preparation of a pharmaceuticalcomposition for preventing or treating tumor growth or formation oftumor metastases.

[0055] RGM (naturally isolated or recombinantly produced) and/orfunctional fragments thereof may be employed for the preparation of apharmaceutical composition for the treatment of neoplastic disorders, inparticular of disorders related to tumor (cell) migration, metastasisand/or tumor invasion. Furthermore, it is envisaged that RGM inhibitsundesired neovascularisation. Said neovascularisation, as an angiogenicdisorder during neoplastic events, should be prevented in order tolimit, inter alia, tumor growth.

[0056] Growth cones of neurons and (invasive) tumor cells secrets acocktail of proteases (uPA, tPA, MNPs, etc.) in order to degradeextracellular matrix. Furthermore, similar mechanisms for adhesion and(cell) migration are employed by these cellular systems. RGM and/or itsfunctional fragments may be employed to actively stimulate withdrawal oflamellipodia of tumor cells and/or to induce their collapse. Asdemonstrated in the appended examples RGM also influences tumor growthbehaviour, i.e. is capable of negatively influencing tumor growth.

[0057] In addition the invention provides for the use of a RGMpolypeptide as defined herein or of a functional fragment or derivativethereof or of a polynucleotide encoding said polypeptide or fragment orderivative for the preparation of a pharmaceutical composition forpreventing, alleviating or treating diseases or conditions associatedwith the activity of autoreactive immune cells or with overactiveinflammatory cells. Most preferably these cells are T-cells.

[0058] In yet another embodiment, the invention provides for the use ofa RGM polypeptide having or comprising, inter alia, the amino acidsequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment orderivative thereof or of a polynucleotide encoding said polypeptide orfragment or derivative for the preparation of a pharmaceuticalcomposition for the treatment of inflammation processes and/orallergies, for wound healing or for the suppression/alleviation of scarformation. Scar tissue is formed by invading cells, most importantly byfibroblasts and/or glial cells. Migration and adhesion of these cellsare required to get to the lesion side. RGM or an activefragment/derivative could prevent accumulation of these cells in thelesion side, thereby preventing or slowing down scar formation. Ininflammatory reactions cells migrate to the inflamed region and RGM orits active fragment/derivative prevent or reduce migration of thesecells to the side of inflammation, thereby preventing overactiveinflammatory reactions.

[0059] In context of the present invention, the term “pharmaceuticalcomposition” also comprises optionally further comprising an acceptablecarrier and/or diluent and/or excipient. The pharmaceutical compositionof the present invention may be particularly useful in preventing and/ortreating pathological disorders in vertebrates, like humans. Saidpathological disorders comprise, but are not limited to, neurological,neurodegenerative and/or neoplastic disorders as well as disordersassociated with seizures, e.g. epilepsy. These disorders comprise, interalia, Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis (FALS/SALS), ischemia, stroke, epilepsy, AIDS dementia andcancer. The pharmaceutical composition may also be used for prophylacticpurposes. Examples of suitable pharmaceutical carriers are well known inthe art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical, intradermal, intranasal or intrabronchialadministration. However, it is also envisaged that the pharmaceuticalcompositions are directly applied to the nervous tissue. The dosageregimen will be determined by the attending physician and clinicalfactors. As is well known in the medical arts, dosages for any onepatient depends upon many factors, including the patient's size, bodysurface area, general health, age, sex, the particular compound to beadministered, time and route of administration, and other drugs beingadministered concurrently. Pharmaceutically active matter may be presentpreferably, inter alia, in amounts between 1 ng and 1000 mg per dose,more preferably in amounts of 1 ng to 100 mg however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, itshould also be in the range of 1 μg to 10 mg units per kilogram of bodyweight per minute, respectively. Progress can be monitored by periodicassessment. The compositions of the invention may be administeredlocally or systemically. Administration will generally be parenterally,e.g., intravenously. The compositions of the invention may also beadministered directly to the target site, e.g., by biolistic delivery toan internal or external target site or by catheter to a site in anartery. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents, depending on the intended use ofthe pharmaceutical composition. Such agents may be drugs acting on thecentral nervous system as well as on small, unmyelinated sensory nerveterminals (like in the skin), neurons of the peripheral nervous systemof the digestive tract., etc.

[0060] It is also understood that the pharmaceutical composition asdefined herein may comprise nucleic acid molecules encoding RGMs (and/orfunctional fragments or derivatives thereof) or corresponding RGMinhibitors or defined herein. As mentioned herein-above, said inhibitorscomprise, but are not limited to, antibodies, aptamer, RGM-interactingpeptides as well as inhibitors interacting with the RGM-encodingpolynucleotides.

[0061] Accordingly, the present invention also provides for a method oftreating, preventing and/or alleviating pathological disorders andconditions as defined herein, whereby said method comprisesadministering to a subject in need of such a treatment a pharmaceuticalcomposition/medicament as defined herein. Preferably, said subject is ahuman.

[0062] The nucleic acid molecules may be particularly useful in genetherapy approaches and may comprise DNA, RNA as well as PNA. Saidnucleic acid molecules may be comprised in suitable vectors, eitherinter alia, gene expression vectors. Such a vector may be, e.g., aplasmid, cosmid, virus, bacteriophage or another vector used e.g.conventionally in genetic engineering, and may comprise further genessuch as marker genes which allow for the selection of said vector in asuitable host cell and under suitable conditions.

[0063] Furthermore, the vectors may, in addition to the nucleic acidsequences encoding RGM or its corresponding inhibitiors, compriseexpression control elements, allowing proper expression of the codingregions in suitable host cells or tissues.

[0064] Such control elements are known to the artisan and may include apromoter, translation initiation codon, translation and insertion sitefor introducing an insert into the vector. Preferably, the nucleic acidmolecule of the invention is operatively linked to said expressioncontrol sequences allowing expression in (eukaryotic) cells.Particularly preferred are in this context control sequences which allowfor correct expression in neuronal cells and/or cells derived fromnervous tissue.

[0065] Control elements ensuring expression in eukaryotic cells are wellknown to those skilled in the art. As mentioned above, they usuallycomprise regulatory sequences ensuring initiation of transcription andoptionally poly-A signals ensuring termination of transcription andstabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers, and/ornaturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in for example mammalian hostcells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter(Rous sarcoma virus), human elongation factor 1α-promoter, CMV enhancer,CaM-kinase promoter or SV40-enhancer. For the expression for example innervous tissue and/or cells derived therefrom, several regulatorysequences are well known in the art, like the minimal promoter sequenceof human neurofilament L (Charron, J. Biol. Chem 270 (1995),25739-25745). Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3(In-Vitrogene, as used, inter alia in the appended examples), pSPORT1(GIBCO BRL) or pGEMHE (Promega), Beside the nucleic acid moleculesdefined herein, the vector may further comprise nucleic acid sequencesencoding for secretion signals. Such sequences are well known to theperson skilled in the art. Furthermore, depending on the expressionsystem used leader sequences capable of directing theprotein/(poly)peptide to a cellular compartment may be added to thecoding sequence of the nucleic acid molecules of the invention and arewell known in the art. The leader sequence(s) is (are) assembled inappropriate phase with translation, initiation and terminationsequences, and preferably, a leader sequence capable of directingsecretion of translated protein, or a part thereof,

[0066] As mentioned herein above, said vector may also be, besides anexpression vector, a gene transfer and/or gene targeting vector. Genetherapy, which is based on introducing therapeutic genes into cells byex-vivo or in-vivo techniques is one of the most important applicationsof gene transfer. Suitable vectors, vector systems and methods forin-vitro or in-vivo gene therapy are described in the literature and areknown to the person skilled in the art; see, e.g., Giordano, NatureMedicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919;Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, CurrentOpinion in Biotechnology 7 (1996), 635-640 Verma, Nature 389 (1997),239-242 WO 94/29469, WO 97/00957, U.S. Pat. No. 5,580,859, U.S. Pat. No.589,66 or U.S. Pat. No. 4,394,448 and references cited therein.

[0067] In particular, said vectors and/or gene delivery systems are alsodescribed in gene therapy approaches in neurological tissue/cells (see,inter alia Blömer, J. Virology 71 (1997) 6641-6649) or in thehypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20 (1999),296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404).

[0068] Further suitable gene therapy constructs for use in neurologicalcells/tissues are known in the art, for example in Meier (1999), J.Neuropathol. Exp. Neurol. 58, 1099-1110. The nucleic acid molecules andvectors of the invention may be designed for direct introduction or forintroduction via liposomes, viral vectors (e.g. adenoviral, retroviral),electroporation, ballistic (e.g. gene gun) or other delivery systemsinto the cell. Additionally, a baculoviral system can be used aseukaryotic expression system for the nucleic acid molecules describedherein.

[0069] The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of partially or completely curing a disease and/oradverse effect attributed to the disease. The term “treatment” as usedherein covers any treatment of a disease in a mammal, particularly ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e. arresting itsdevelopment; or (c) relieving the disease, i.e. causing regression ofthe disease.

[0070] In yet another embodiment, the present invention provides for theuse of a (RGM) polypeptide and/or a polypeptide having or comprising theamino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functionalfragment or derivative thereof or of a polynucleotide encoding saidpolypeptide or fragment or derivative as a marker of stem cells. Sinceit is envisaged that stem cells as well as their undifferentiatedprogenitor cells express RGM, RGM and/or functional fragments orderivatives thereof may be employed to influence thedifferentiation/differentiation pattern of said stem cells.

[0071] It is furthermore envisaged that antibodies directed againstRGMs, in particular directed against polypeptides disclosed herein orcomprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 (or(a) functional fragment(s)/derivative(s) thereof) may be employed toinfluence the differentiation of (neuronal) stem cells and (neuronal)progenitor cells. It is particularly preferred that said antibodies (aswell as other RGM-inhibitors and/or RGM-binding molecules) be employedto selectively label stem cells. Therefore these reagents may beemployed as markers for stem cells. It is also envisaged that peptidesor derivatives be employed in said purpose.

[0072] In a particularly preferred embodiment of the present invention,the polypeptide and/or fragment thereof which comprises or has an aminoacid sequence as depicted in SEQ ID NOs 18, 20, 23 or 25 and/or is a RGMmolecule to be used in accordance with their invention is a soluble,i.e. not membrane bound molecule.

[0073] As shown in Davis (1994), Science 266, 816-819 ephrins, inparticular A-ephrins, are not active in soluble, monomeric form. Incontrast, soluble RGMs are active and may function without anymembrane-attachment RGM, in contrast to ephrins, is capable ofself-formation of dimers and/or of the formation of higher aggregates.The invention also provides for the use of a RGM molecule and/or apolypeptide having or comprising the amino acid sequence of SEQ ID NOs18, 20, 23 or 25 or of a functional fragment or derivative thereof or ofa polynucleotide encoding said polypeptide or a fragment or a derivativefor the preparation of a pharmaceutical composition for alleviating,preventing and/or treating homeostatic and/or bleeding disorders and/orvascular damage.

[0074] It is envisaged, without being bound by theory, that RGMs may,due to their structural homology to von-Willebrand factor (vWF), beemployed in the treatment of said disorders/diseases. Furthermore, it isenvisaged that RGM may interact with von-Willebrand factor and that saidmolecule, thereby, influences the activity of vWF. Furthermore, theinhibitors as defined herein should be employed in disorders whereimmune cells invade the brain, like multiple sclerosis,encephalomyelitis disseminata.

[0075] The present invention also provides for the use of an antibody ora fragment or a derivative thereof, or an aptamer, or a binding moleculecapable of interacting with a polypeptide having or comprising the aminoacid sequence of SEQ ID NOs 18, 20, 23 or 25 or with functional fragmentor derivative thereof or of an nucleid acid molecule capable ofinteracting with a polynucleotide encoding said polypeptide or afragment thereof for the preparation of a diagnostic composition fordetecting neurological and/or neurodegenerative disorders ordispositions thereto.

[0076] The diagnostic composition may be used, inter alia, for methodsfor determining the expression of the nucleic acids encoding RGMpolypeptides by detecting, inter alia, the presence of the correspondingmRNA which comprises isolation of RNA from a cell, contacting the RNA soobtained with a nucleic acid probe as described above under hybridizingconditions, and detecting the presence of mRNAs hybridized to the probe.Furthermore, corresponding mutations and/or alterations may be detected.Furthermore, RGM (poly)peptides can be detected with methods known inthe art, which comprise, inter alia, immunological methods, like, ELISAor Western blotting.

[0077] The diagnostic composition of the invention may be useful, interalia, in detecting the prevalence, the onset or the progress of adisease related to the aberant expression of a RGM polypeptide.Accordingly, the diagnostic composition of the invention may be used,inter alia, for assessing the prevalence, the onset and/or the diseasestatus of neurological, neurodegenerative and/or inflammatory disorders,as defined herein above. It is also contemplated that anti-RGMantibodies, aptamers etc. and compositions comprising such antibodies,aptamers, etc. may be useful in discriminating (the) stage(s) of adisease.

[0078] The diagnostic composition optionally comprises suitable meansfor detection. The nucleic acid molecule(s), vector(s), antibody(ies),(poly)peptide(s), described above are, for example, suitable for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. Examples of well-known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of theinvention.

[0079] Solid phase carriers are known to those in the art and maycomprise polystyrene beads, latex beads, magnetic beads, colloid metalparticles, glass and/or silicon chips and surfaces, nitrocellulosestrips, membranes, sheets, duracytes and the walls of wells of areaction tray, plastic tubes or other test tubes. Suitable methods ofimmobilizing nucleic acid molecule(s), vector(s), host(s),antibody(ies), (poly)peptide(s), fusion protein(s) etc. on solid phasesinclude but are not limited to ionic, hydrophobic, covalent interactionsand the like. Examples of immunoassays which can utilize said compoundsof the invention are competitive and non-competitive immunoassays ineither a direct or indirect format. Commonly used detection assays cancomprise radioisotopic or non-radioisotopic methods. Examples of suchimmunoassays are the radioimmunoassay (RIA), the sandwich (immunometricassay) and the Northern or Southern blot assay. Furthermore, thesedetection methods comprise, inter alia, IRMA (Immune RadioimmunometricAssay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay),FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent ImmuneAssay). Furthermore, the diagnostic compounds of the present inventionmay be are employed in techniques like FRET (Fluorescence ResonanceEnergy Transfer) assays.

[0080] The nucleic acid sequences encoding RGMs of other species as wellas variants of RGMs are easily deducible from the information providedherein. These nucleic acid sequences are particularly useful, as pointedout herein above, in medical and/or diagnostic setting, but they alsoprovide for important research tools. These tools may be employed, interalia, for the generation of transgenic animals which overexpress orsuppress RGMs or wherein the RGM gene is silenced and/or deleted.Furthermore, said sequences may be employed to detect and/or ellucidateRGM interaction partners and/or molecules binding to and/or interferingwith RGMs.

THE FIGURES SHOW

[0081]FIG. 1: RGM protein fractions Induce collapse of RGC growth cones.

[0082] Solubilized membrane proteins from E9/E10 chick brains wereloaded on two different ion exchange columns, a DEAE anion exchangecolumn and a cation excange column. RGM was eluted from the cationexchange column at a NaCl concentration of 200-400 mM in two 1 mlfractions (4+5) was incorporated into lecithin vesicles and lecithinvesicles were used in collapse experiments with RGC growth cones.RGM-containing fractions (4+5, arrows), but not RGM-free fractionsinduced extensive collapse (>90%) of RGC growth cones. Neither ephrin-A5nor ephrin-A2 could be detected with specific antibodies, inRGM-fractions. RGC axons and growth cones on laminin were stained withAlexa-Phalloidin. Western blots from two dimensional gels were incubatedwith the F3D4 monoclonal antibody, and were subsequently stained by awhole protein, india ink stain.

[0083]FIG. 2: Comparative two dimensional gel analysis of tectalproteins and RGM sequences.

[0084] A: Membranes from E9/10 anterior and posterior chick tecta wereenriched and treated with buffer (C) or with PI-PLC (E), to removeGPI-anchored proteins. The putative RGM (arrow inAnterior-E+Posterior-E), a PI-PLC cleavable basic protein with amolecular weight of 33 kDa, was cut out and was used fornanoelectrospray tandem mass spectrometry. Two dimensional gels werestained with silver. No anterior-posterior difference of the RGMcandidate is observed in these gels, this is probably due to thepresence of two other proteins in the selected spot. B: Deduced RGMpeptide sequences

[0085]FIG. 3: Nucleotide and amino acid sequence of RGM.

[0086] A. Nucleotide sequence of RGM.

[0087] B. Amino acid sequence of RGM. Peptides derived frommicrosequencing are highlighted in bold and peptides used for makingpolyclonal antibodies are underlined. Potential N-glycosylation sitesand an RGD tripeptide, potential cell attachment site are underlined byasterics.

[0088] C. Schematic view of the RGM protein. Hydrophobic domains arpresent a the N- and C-termini of the protein. Epitopes of the twopolyclonal anti-RGM antibodies are demarcated.

[0089]FIG. 4: The polyclonal and the monoclonal RGM antibody recognizethe same 33 kDa protein.

[0090] A. The anti-RGM1 antibody binds to a GPI-anchored CRD-(crossreacting determinant) positive 33 kDa protein. Left blot: An anti-CRDantibody binds to a low abundant, 33 kDa protein (arrow), present in theE (PI-PLC supernatant) but not the C fraction (control supernatant).Right blot: Anti-RGM1 staining of a GPI-anchored 33 kDa protein on awestern blot with supernatant from E9/E10 chick brain membranes.

[0091] B. The GPI-anchored 33 kDa antigen of the anti-RGM1 antibody ismore abundant in posterior (pos.) than in anterior (ant.) tectalmembranes. Left blot: rabbit preimmune serum did not bind to any proteinon western blots with PI-PLC supernatant protein from anterior andposterior tectum. Right blot: Anti-RGM1 binding to a 33 kDa protein.E=PI-PLC supernatant from tectal membranes, C=control supernatants fromtectal membranes.

[0092] C. Anti-RGM1 and F3D4 recognize the same antigens in tectalmembranes. Left blot: F3D4 staining of tectal membrane proteins. Adouble band at 33 kDa (lower arrow) and a hardly visible band at 35 kDa(upper arrow) are recognized. Right blot: Anti-RGM1 staining reveals thesame staining pattern with 33 and 35 kDa antigens (arrows). Contrary tothe membrane fraction, where 3 different protein bands are observed,only one band is detected in most western blots with PI-PLCsupernatants.

[0093] For detection on western blots, a secondary, alkalinephosphatase—conjugated antibody was used and NBT (nitro bluetetrazolium) and BCIP (bromochloroindolyl phosphat) was used for thecolour reaction.

[0094]FIG. 5: The RGM anti-sense probe hybridizes to an mRNA with gradedexpression along the anterio-posterior axis.

[0095] A, B: RGM-mRNA is expressed in a periventricular gradient in thetectum of an E9 chick embryo. In a more superficial layer (arrows), RGMis also expressed but at much lower level. The anterior tectal pole isto the right, the posterior to the left.

[0096] C, D: No staining is detected with the RGM sense probe, onparallel cryostat sections from E9 chick tecta. The anterior tectal poleis to the right, the posterior to the left.

[0097]FIG. 6: Recombinant RGM induces collapse of retinal growth cones.

[0098] A: RGC axons were grown on laminin-coated coverslips and affinitypurified recombinant RGM was added at a final concentration of 10 ng/ml.More than 90% of temporal retinal growth cones are collapsed.

[0099] B: Neighboring, RGM—free fractions from affinity purification didnot induce collaps of temporal growth cones. Supernatants from cos-7cells transfected with an empty plasmid, did not possess anycollapse-inducing activity (data not shown).

[0100] In A and B, retinal axons and growth cones were stained with theF-actin stain Alexa-Phalloidin.

[0101]FIG. 7: Recombinant RGM guides temporal retinal axons in thestripe assay.

[0102] A, B: Temporal retinal axons avoid the RGM—containing stripes(demarcated with red flourescent beads). Membranes from RGM—transfectedcos-7 cells (marked with beads) and anterior tectal membranes were usedto prepare striped carpets.

[0103] C, D: Temporal retinal axons do not show any avoidance recation,when membranes from cos-7 cells, transfected with an empty plasmid (redbeads) were used.

[0104] In A-D, striped membrane carpets were in addition coated withlaminin to enhance retinal axon growth in accordance with a previousprotocol (Monschau et al. 1997).

[0105]FIG. 8: RGM staining in endothel of (human) brain.

[0106] RGM immunoreactivity was detected in endothelial and vascularsmooth muscle cells (SMC), both, in healthy, neuropathological unalteredcontrol brains and injured brains, suggesting a constitutive,physiological role in vascular homeostasis.

[0107]FIG. 9: RGM expression in a lesion of a human being deceased dueto severe brain injury (1-2 hours after his death). RGM expression oninfiltrating cells from the immune system.

[0108] Upregulation of cellular RGM expression correlated with the timecourse and appearance of infiltrating leukocytes and activation ofmicroglia/macrophages after injury (Stoll et al., 1998).

[0109] Early after injury (up to 2.5 days), RGM immunoreactivity wasfound on leukocytes of granulocytic, monocytic and lymphocytic origin invessels within ischemic tissue. Paralleled by edema formation, up to 1-7days, RGM-positive cells were found extravasating outside the vascularwalls into the focal ischemic lesioned parenchyma. In perivascularregions, RGM-positive cells formed clusters in the Virchow-Robin spacesfrom day 1-7, which subsided later. These peri-vascular cells, alsoreferred to as adventitial or perithelial cells are characteristicallyalert immune cells (Kato and Walz, 2000; Streit et al., 1999).

[0110]FIG. 10: RGM expression in a brain lesion (human).

[0111] With increasing time after brain injury, most remarkable changescorresponded to areas of ongoing scar formation. In these areas, welldefined extracellular RGM-positive laminae and RGM-positivefibroblastoid and reactive astrocytic cells were visible condensingadjacent to the border zone. These RGM-positive laminae increased inmagnitude and regional extend over time.

[0112] The Examples illustrate the invention.

EXAMPLE I Microsequencing of an RGM Candidate

[0113] To separate RGM from the A-ephrins, a combination of twodifferent ion exchange columns was employed. RGM, in contrast to theA-ephrins, bound to a strong cation exchanger and was eluted at a saltconcentration of 200-400 mM NaCl. After incorporation of RGM intolecithin vesicles, strong collapse-inducing activity was observed inRGM-fractions (fractions 4+5, FIG. 1) but not in neighboring RGM-freefractions (fraction 6, FIG. 1). Neither ephrin-A5 nor ephrin-A2 waspresent in these fractions, proving thereby that RGM function does notrequire presence of the A-ephrins.

[0114] To get peptide sequences from RGM, microsequencing of allproteins, (cleaved from the membrane by treatment with the enzyme PI-PLCand having a molecular weight of 30-35 kDa and an isoelectric pointbetween 7 and 9 was carried out). To this aim, anterior and posteriormembranes from embryonic chick tecta (E9/10) were prepared with somemodifications as described previously (Walter, Development 101, (1987),685-96) and membrane pellets were subject to treatment with enzymePI-PLC (E fraction) or buffer alone (C fraction). In particular,Membranes from embryonic chick tecta (E9/10) were prepared with somemodifications as described previously (Walter et al., 1987). All stepswere performed at 4° C. Tecta from 100 chick embryos were isolated andwere divided into three parts equal in length along theanterior-posterior axis. The middle tectal parts were discarded and theanterior and posterior parts were worked up separately. Membranes werewashed with PBS containing protease inhibitors and were centrifuged.Tectal membrane pellets were resuspended in triethanolamin buffer andwere treated with the enzyme PI-PLC (50 mU Boehringer Mannheim/RocheDiagnostics GmbH), to remove glycosylphosphatidylinositol-anchored(GPI-anchored) proteins from the membranes. No PI-PLC was added to theother anterior and posterior tectal membrane fractions, thecontrol-fractions (C). Enzyme (E) and control (C) fractions wereincubated at 37° C. for 1.5 hours and membrane suspensions werecentrifuged at 400.000×g in a Beckmann TLA 100.3 rotor. Supernatantswere collected and their protein concentrations were determined(Bradford 1976, modified by Zor and Selinger, 1996). Supernatants wereprecipitated with ice cold 10% trichloroacetic acid, were centrifugedand protein pellets were washed in ethanol-ether (1:1 v/v) andsolubilized in sample buffer (8.5 M urea, 5% β-mercaptoethanol, 2.5%ampholytes pH 3-10, 2% NP 40).

[0115] E fractions and C fractions were loaded onto two dimensionalgels, and after silver staining candidate proteins in the E-fractions(FIG. 2A, arrows) were cut out and subject to in gel tryptic digestionand nanoelectrospray ionization (Wilm, Nature 379 (1996), 466-9).

[0116] In detail, said 2D gelelectrophoresis and the protein sequenceanalysis was carried out as outlined herein below:

[0117] Tectal proteins resuspended in sample buffer, were separatedusing two dimensional gel electrophoresis. 20 μg of tectal protein wasloaded on each gel. Non-equilibrium pH gradient electrophoresis (NEPHGE)followed by SDS-PAGE in the second dimension was performed as describedby Boxberg (1988). After the SDS PAGE, gels were stained by a modifiedsilver staining protocol from Heukeshoven and Demick (Heukeshoven &Demick, Electrophoresis 9, (1988), 372-375).

[0118] Silver-stained proteins in the 2D gels, with a basic isoelectricpoint and a molecular weight of 33/35 kDa, present in the PI-PLC treatedE fraction but not in the C fraction, were cut out using a sharp andsterile scalpel.

[0119] Microsequencing was done using the technique of nano-electrospraytandem mass spectrometry as previously described (Wilm et al., 1996).The protein spots were digested in gel by trypsin and the resultingpeptides were adsorbed and stepwise eluted into the electrospray sourcefor mass spectral analysis. Nanoelectrospray was performed on an API III(Perkin-Elmer) mass spectrometer as described by Wilm and Mann (Wilm &Mann, Anal. Chem. 68 (1996), 1-8). After selecting an ionized peptidefrom the peptide mixture, the peptide was fragmented and the peptidefragments were analysed.

[0120] Mass spectrometric microsequencing of ionized peptides from thespot marked by arrows in FIG. 2, yielded ten different peptides, withlengths of 5-14 amino acids as shown in FIG. 2B; (SEQ ID NOs 1-10). Theselected spot, was present in anterior and posterior PI-PLCsupernatantes at similar levels. RGM is however more abundant inposterior than in anterior tectal membranes and the disappearance of theap-difference in the 2D-gels was most likely caused by two differentproteins unrelated to RGM and present in the selected spot.

EXAMPLE II Cloning of the RGM Gene

[0121] Three out of the ten peptide sequences (SEQ ID NOs 1 to 10)obtained by nanoelectrospray tandem mass spectrometry were used forsynthesis of degenerate oligonucleotide primers and PCR experiments wereperformed as follows: Three out of the ten peptide sequences obtained bynanoelectrospray tandem mass spectrometry were used for synthesis ofdegenerate oligonucleotide primers and their complementary sequences.P1F: 5′-ATGCC(AGCT)GA(AG)GA(AG)GT(AGCT)GT(AGCT)-3′ (SEQ ID NO:11) P1R:5′-TT(AGCT)AC(AGCT)AC(CT)TC(CT)TC(AGCT)GGCAT-3′ (SEQ ID NO:12) P2F:5′-GA(CT)AC(AGCT)TT(CT)CA(AG)AC(AGCT)TG(CT)AA-3′ (SEQ ID NO:13) P2R:5′-TT(AG)CA(AGCT)GT(CT)TG(AG)AA(AGCT)GT(AG)TC-3′ (SEQ ID NO:14) P3F:5′-AA(CT)CA(AG)CA(AG)(CT)T(AGCT)GA(CT)TT(CT)CA-3′ (SEQ ID NO:15) P3R:5′-TG(AG)AA(AG)TC(AGCT)A(AG)(CT)TG(CT)TG(AG)TT-3′ (SEQ ID NO:16)

[0122] Moloney murine leukemia virus reverse transcriptase and randomhexamer primers were used to synthesize single-stranded cDNA from E9chick tectum total RNA. Combinations of forward (F) and reverse (R)primers were added to the cDNA and PCR amplification was done using Taqpolymerase. The following PCR conditions were used: an initialdenaturation step at 95° C. for 5 min followed by 30 cycles of 95° C.for 40 s, 50° C. for 1 min, 72° C. for 2 min. The PCR products werecloned into the pGEM T vector (Promega) and four positive clones weresequenced using the ALF express sequencer (Pharmacia). The sequenceyielded an ORF, containing most of the peptide sequences obtained bymicrosequencing. The 459 bp fragment was used for screening a cDNAlibrary to obtain the full length sequence and for further analysis suchas Northern bloting and in situ hybridization.

[0123] The PCR products were loaded onto agarose gels stained withethidium bromide and a PCR product of 459 bp in length, was obtained andcloned Into the pGEM T vector. After sequencing, most of the peptidesequences were found in the PCR product, confirming that the correctcandidate was amplified. The 459 bp fragment was used for screening anE14 chicken brain cDNA library. Positive clones contained an insert ofapproximately 4 kb and sequencing confirmed the presence of the 459 bpfragment and additional downstream sequences, including a stop codon.Upstream sequences were obtained by performing 5′-RACE.

[0124] In detail, the 459 bp probe was used to screen 500.000 plaques ofan E14 chicken brain library, cloned in the λ Zap vector. After twoscreening rounds, eight single plaques were isolated and the relatedinserts were cloned into the Bluescript vector using the rapid excisionkit (Stratagene). The positive clones, analysed by restrictiondigestions, contained an insert of approximately 4 kb and sequencingconfirmed the presence of the 459 bp fragment and additional downstreamsequences, including a stop codon. To get the sequence of the regionupstream of the 459 bp fragment, a 5′-RACE was performed according tothe manufacturer's protocol using the RACE kit from Boehringer Mannheimand total RNA from E9 chick tecta. A 700 bp band was amplified,purified, cloned into pGEM T vector, and 5 positive clones weresequenced. The sequence had an ORF with two methionines which could actas potential start sites. The full length sequence of RGM was confirmedindependently several times.

[0125] For in situ and Northern blot experiments, the 459 bp fragmentwas cloned into the Bluescript KS vector (Stratagene) and anti-sense andsense probes were produced by using the SP6 and T7 polymerases,respectively.

[0126] This 5′-RACE yielded an ORF with two methionines, potential startsites. The complete ORF of RGM is 1302 nucleotides in length and encodesa protein consisting of 434 amino acids (FIG. 3A; SEQ ID NO:17). Twohydrophobic domains are present at the N-terminus and C-terminus,respectively (FIG. 3B; SEQ ID NO:18), and two different algorithmssuggested that the N-terminal hydrophobic domain encodes a signalpeptide (best cleavage site predicted: at aa 29), the C-terminal domain,a GPI-anchor domain (best cleavage site predicted: at aa 406). RGM hasno significant homology to any other protein, present in the databasesand does not carry any specific domain or motif, except an triamino acidmotif, the RGD site, a potential cell attachment site (Ruoshlahti, Annu.Rev. Cell Dev. Biol. 12 (1996), 697-715). Preliminary results suggestthat this site is dispensable for RGM function. Polyclonal antibodies,named anti-RGM1 (against aas: 276-293) and anti-RGM2 (against aas:110-130), raised against two peptides of the recombinant RGM molecule,recognize a GPI-anchored 33 kDa molecule, which is present at higherlevels in posterior than in anterior tectal membrane PI-PLC supernatants(FIG. 4A). In membrane fractions at least three protein bands appear, adouble band at 33 kDa and a single band at 35 kDa. These protein bandsare recognized by the polyclonal anti-RGM1 antibody and the monoclonalF3D4 antibody (Müller (1996), loc. cit) (FIG. 4B). Both antibodies showidentical staining patterns on western blots and immunoprecipitationexperiments with ant-RGM1 resulted in pull down of a GPI-anchored,F3D4-positive protein. These results prove, that the antigens of theF3D4 monoclonal antibody and of the anti-RGM1 polyclonal antibody areidentical.

[0127] RGM is the first member of a new class of axon guidancemolecules, sharing no sequence homology with ephrins, netrins, slits,semaphorins and any other axon guidance molecules.

[0128] The corresponding human RGM sequence (SEQ ID NO:20) could bededuced by screening the human genome database with the deduced chickenRGM sequence.

EXAMPLE III RGM mRNA is Expressed in a Gradient in the Optic Tectum

[0129] To analyse expression of RGM-mRNA in the tectum opticum, an RGManti-sense probe was used in in situ hybridization experiments oncryostat sections from E9 chick tecta. Strongest staining is observed inthe periventricular layer, surrounding the tectal ventricle and stainingintensity is much stronger in posterior tectum than in anterior tectum(FIGS. 5A, B). Cell bodies of radial glial cells are located in theperiventricular layer and the staining pattern confirms previous datausing the monoclonal F3D4 antibody, where staining of glial endfeet andof glial cell bodies was observed (Mueller; (1996), loc. cit.; Mueller,(1997), loc. cit.). In a more superficial layer, a much weaker stainingis detected with the RGM anti-sense probe but a differential expressionbetween anterior and posterior tectal poles is hard to detect in thislayer. In this layer tectal neurons are RGM-positive. This is in linewith the expression of RGM by a subpopulation of tectal neurons.Overall, the staining pattern with the RGM anti-sense probe looks verysimilar to the expression pattern of ephrin-A5 with both messages beingfound in a periventricular and in a more superficial tectal layer. Nostaining is detectable with the RGM-sense probe.

[0130] On northern blots with tectal RNA, the RGM anti-sense probemarked two transcripts at 5.5 and 6.1 kb. Both messages aredown-regulated at E14 with the smaller message being no longerdetectable and the larger transcript being clearly present, albeit atlower levels.

[0131] RGM is active in in vitro assays and shows a graded expression inthe tectum opticum of vertebrates. Based on Southern blot data it isassumed that there are least two additional family members which mighthave similar guidance activity. (see FIG. 11)

EXAMPLE IV Recombinant RGM is Active in Collapse and Stripe Assay

[0132] To analyse the function of recombinant RGM, the full length RGMcDNA was used to transfect cos-7 cells with a lipofection procedure. Thefull length RGM cDNA was cloned into the KpnI site of the expressionvector pTriEx-1 (Novagen). Cos-7 cells were transfected with thepTriEx-1 plasmid containing RGM cDNA or with the empty plasmid using theSuperfect transfection reagent (Qiagen) according to the manufacturer'sprotocol. The DNA-Superfect mixture was added to Cos-7 cells growing in10 cm dishes. 2 hours later medium was removed, cells were washed withPBS and grown for an additional 48 hours in fresh medium. Conditionedmedium was collected, run over an RGM-affinity column and RGM-containingfractions and RGM-free control fractions were directly used in collapseassay experiments. For stripe assay experiments, RGM-transfected Cos-7cells and empty plasmid transfected cells were washed with PBS andharvested using a rubber policeman in the presence of homogenizationbuffer containing protease inhibitors. Conditioned medium of cos-7 cellstransfected with the RGM-pTriEx-1 plasmid was collected and run over ananti-RGM1 antibody column. Eluted fractions were evaluated with asensitive and rapid dot blot assay and RGM-positive fractions were addedto retinal axons growing on a laminin substratum. At a finalconcentration of 10 ng/ml, soluble RGM induced collapse of 90% oftemporal RGC growth cones (FIG. 6A). Neighboring, RGM-free fractions orconditioned and concentrated supernatants from cos-7 cells transfectedwith the empty plasmid did not possess any collapse-inducing activity(FIG. 6B). Recombinant RGM is active in soluble form, is a strongdifference between RGM and the A-ephrins and suggests a role for achemotropic mechanism, in etablishing the retinotectal map.

[0133] For preparation of striped membrane carpets, membranes from RGM-or mock-transfected cells were used. Carpets consisting of alternatelanes of membranes from mock-transfected cos-7 cells and fromRGM-transfected cells were offered to temporal and nasal RGC axons. Toenhance the poor outgrowth-stimulating activity of cos-7 membranes,anterior tectal membranes or laminin were added. Collapse assay andstripe assays were prepared and employed as follows: The collapse assaywas performed as descibed (Cox, (1990), loc. cit.; Wahl, J. Cell Biol.149(2) (2000), 263-70). 5 μl of the RGM-positive fraction from theRGM-cos supernatant or supernatant from control cos cells or RGM-freefractions, was added to the retinal cultures. One hour later cultureswere fixed by carefully adding 1 ml of fixative (4% paraformaldehyde,0.33 M sucrose, pH 7.4). 4-12 hours later, cultures were washed andstained by Alexa-Phalloidin (Molecular Probes), following therecommendations of the manufacturer. Stained cultures were stored on acomputer using a CCD camera and the images were analysed with the SISanalysis imaging software.

[0134] Stripe assay experiments were performed as previously describedby Walter et al. (1987). Membrane carpets consisted of lanes of anteriortectal membranes mixed with membranes from RGM-transfected cos cells(ratio: 1:1), alternating with lanes consisting of anterior membranesmixed with membranes from empty plasmid transfected cos cells (ratio1:1). In an alternative protocol, membrane carpets consisting ofalternating lanes of membranes from RGM-transfected cos cells and ofcontrol cos membranes, were incubated for 2 hours at 37° C. with 20μg/ml laminin (Becton-Dickinson). Before use, the carpets were washedwith Hank's buffer (2×).

[0135] On these carpets, temporal RGC axons, but not nasal axons, showeda clear repulsive avoidance behaviour, growing on the RGM-free membranestripes (FIGS. 7A-D). These results demonstrate, that the recombinantRGM protein is not only active in collapse but also in stripe assays.

[0136] RGM shares with the A-ephrins A2 and A5 the GPI-anchor, thegraded expression and functional activity in two different in vitrosystems. Its activity is however different from the two A-ephrins inother respects. The specificity of its activity is not restricted totemporal axons and growth cones. Nasal axons and growth cones alsoreact, albeit at higher RGM concentrations. This is in line with theprevious observations, that temporal retinal axons react more stronglyto RGM than nasal retinal axons (Stahl, (1990), loc.cit). For ephrin-A5,a slight difference in sensitivity of temporal and nasal retinal axonshas been observed, this difference is however not as pronounced as withRGM (Drescher, Cell 82 (1995), 359-70). Besides the strongerconcentration dependancy of RGM function, another crucial difference isthat RGM, in contrast to both ephrin-A5 and ephrin-A2, seems to beactive in soluble form and apparently does not require aggregation tostimulate its currently unknown retinal receptor. These in vitro resultsunderscore the difference between ephrins and RGM. In the stripe assay,inactivation of RGM using the F3D4 monoclonal antibody and thechromophore-assisted laser inactivation (CALI) method, resulted incomplete neutralization of repulsive guidance activities of posteriortectal membranes in more than 50% of the experiments (Mueller, (1996),loc. cit.) F3D4 however neither binds ephrin-A2 nor ephrin-A5 (Mueller,(1997), loc. cit.) and it was therefore suggested that the A-ephrins andRGM somehow interact in special membrane domains to which they arerecruited by their GPI-anchors. Such a colocalization could explain theresult, that inactivation of RGM lead in addition to inactivation ofephrin-A2 and ephrin-A5 and could explain the complete neutralizationobserved in the stripe assay experiments (Mueller, (1996), loc. cit.).The functional relationship of RGM with ephrin-A2 and ephrin-A5 and thein vivo role of RGM need to be adressed, especially since both ephrinshave been shown to be important molecular determinants for topographicmap formation in vertebrates (Nakamoto, Cell 86 (1996), 755-66; Frisen,Neuron 20 (1998), 235-43; Feldheim, Neuron 21 (1998), 563-74; Picker,Development 126 (1999), 2967-78; Feldheim, Neuron 25 (2000), 563-74;Brown, Cell 102 (2000), 77-88). There are however evidences from twovertebrates, which suggest that others factors, besides the ephrins, arerequired for formation of the retinotectal map. Deletion of either theephrin-A2 or the ephrin-A5 gene in mice, resulted in mapping phenotypeswith some retinal axons forming ectopic termination zones in thesuperior colliculus (SC), the mammalian homologue of the optic tectum,and with nasal retinal axons overshooting the SC and terminating in theinferior colliculus. In ephrin-A2^(−/−) mice, temporal axons showedmapping errors with ectopic termination zones, but nasal axons did notshow any mapping errors in contrast to the ephrin-A5^(−/−) mice whichhad defects in topographic mapping of nasal but not temporal axons(Frisen, (1998), loc.cit.; Feldheim, (2000), loc. cit.). Deletion ofboth genes should therefore result in a much more disturbed mapping ofboth nasal and temporal retinal axons along the anterior-posterior axisof the SC. This is actually observed in double mutant ephrin-A2^(−/−)A5^(−/−) homozygotes but a topographic bias of both nasal and temporalaxons was still present, with the majority of temporal and nasal retinalaxons being confined to their anterior and posterior tectal halfs,respectively (Feldheim, (2000), loc. cit.; Goodhill, Neuron 25 (2000),501-3). These results point to a role of RGM as one of the additionalfactors required for mapping along the anterior-posterior axis. Such arole is supported by the graded anterior-low posterior-high expressionof this molecule in the SC of mammals (Mueller, (1997), loc. cit.).

[0137] The zebrafish mutant acerebella (ace) is mutant in fgf8 and lacksthe midbrain-hindbrain boundary region and the cerebellum (Reifers,Development 125 (1998), 2381-95; Picker, (1999), loc. cit.). As a resultthe tectum is much smaller in ace mutants than in wildtype and theexpression levels of all three zebrafish A-ephrins are changed withephrin-A2 and ephrin-A5a being still expressed at low and anteriorlevels in ace tecta and with ephrin-A5b being completely eliminated(Picker et al., 1999). In ace mutant tecta, mapping of retinal axonsalong the anterior-posterior axis is normal in dorsal tectum and is notcompletely lost in ventral tectum, suggesting the involvement of othergraded guidance cues, not seriously affected by the fgf8 mutation in theace zebrafish mutants (Picker et al., 1999). Dorsoventral patterning inboth zebrafish ace mutants and ephrin-A2^(−/−) A5^(−/−) double knock outmice is affected.

[0138] RGM, with its graded expression along the anterior-posterior axisof the tectum and its ability to function in a secreted andmembrane-coupled way, is an important player for topographic mapformation.

EXAMPLE V

[0139] Materials and Methods

[0140] 1. Patients

[0141] 21 brains of patients with clinical history andneuropathologically confirmed diagnosis of focal cerebral infarctionsand 25 brains of patients with traumatic brain injury were included inthis study. Infarctioned brain tissue was derived from an updated strokeand trauma brain-bank (Table 1,2) reported previously (Postler et al.,1997, Beschorner et al., 2000). Tissue specimen procurement wasperformed according to the ethical guidelines of the University ofTuebingen. Patients with altered immune status because ofimmunosuppressive therapy or meningitis/encephalitis were excluded fromthis study. As controls, the results were compared to tissue fromcorresponding areas of 4 normal non-ischemic brains described previously(Schwab et al., 2000). In addition to patient data, haematoxyline-eosine(HE), luxol fast blue (LFB) and iron (Fe) staining was used forevaluation of the typical histological features defined as standardindication of infarct (Kalimo et al., 1996) and trauma age (Graham andGennarelli, 1996).

[0142] 2 Immunohistochemistry

[0143] After formaldehyde fixation and paraffin-embedding, rehydrated 2μm sections were boiled (in an 600 W microwave oven) seven times for 5min in citrate buffer (2.1 g sodium citrate/liter, pH 7.4). Endogenousperoxsidase was inhibited with 1% H₂O₂ in methanol (1:10; 15 min).Sections were incubated with 10% normal porcine serum (Biochrom, Berlin,FRG) to block non-specific binding of immunoglobulins. Monospecificpolyclonal antibodies directed against RGM were diluted (1:10) in 1% BSA(bovine serum albumin) TBS (Tris-balanced salt solution, containing0.025 M Tris, 0.15 M NaCl) and incubated over night at room temperature.Specific binding of the antibodies were detected with a secondarybiotinylated swine anti-rabbit IgG F(ab)₂ antibody fragment 1:400 for 30min (DAKO, Hamburg, FRG), followed by incubation with a peroxidaseconjugated streptavidin-biotin complex (DAKO, Hamburg, FRG). The enzymewas visualized with diaminobenzidine as a chromogen (Fluka, Neu-Ulm,FRG). Sections were counterstained with Mayer's Hemalaun. Negativecontrols consisted of sections incubated in the absence of the primaryantibody. Specificity of polyclonal RGM antibody was confirmed byinhibition of staining using human ischemic brain tissue afterpre-incubation for 3 h on ice with access of the cognate RGM peptide.

[0144] 3. Double Labeling Experiments

[0145] In double labelling experiments, a cell-type or activationspecific antigen was first labelled using the ABC procedure incombination with alkaline phosphatase conjugates. Specific antigens werelabelled with antibodies against GFAP (glial fibrillary acidic protein,monoclonal, Boehringer Mannheim, Germany, 1:100) to detect astrocytes,MBP (myelin basic protein, polyclonal, oligodendrocytes, Dako, 1:500)and CD68 (Dako, 1:100) for microglia/macrophage identification.Activated microglia/macrophages were detected with antibodies directedagainst HLA-DR, -DP, -DQ (MHC class II, DAKO, Glostrup, Denmark, 1:100)or MRP-8 (8-5C2, BMA, Augst, Switzerland, 1:100) (Postler et al., 1997).Lymphocytic subpopulations were classified with monoclonal antibodiesagainst CD4 (T-helper lymphocytes, 1:10, Dako) and CD8 (Tcytotoxic/suppressor lymphocytes, 1:500, Dako) and CD20 (pan B cellmarker, 1:200, Dako). In order to detect extracellular basal laminastructures in vessels and during scar formation mouse laminin (1:500,Chemicon) antibodies were used and rabbit fibronectin (1:100, Dako)antibodies were used to detect matrix deposition. Furthermore, in orderto characterize the cellular proliferation response, sections wereincubated with the S phase specific PCNA (proliferating cell nuclearantigen, 1:100, Dako) monoclonal antibodies. Briefly, slices weredeparaffinized, irradiated in a microwave oven for antigen retrieval andincubated with non specific porcine serum as described above.Visualization was achieved by adding biotinylated secondary antibodies(1:400) for 30 min and alkaline phosphatase conjugated ABC complexdiluted 1:400 in TBS-BSA for 30 min. Consecutively, slices weredeveloped with Fast-Blue BB salt chromogen-substrate solution yielding ablue reaction product. Between double labelling experiments, slices wereirradiated in a microwave for 5 min in citrate buffer. Then RGM wasimmunodetected as described above.

[0146] 4. Evaluation and Statistical Analysis

[0147] Data were calculated as means of labelled cells (MLC, ±SEM) fromborder zones or remote areas of the same tissue section and werecompared to normal control brains using the two-tailed unpairedstudent's t-test. Border zones were defined as peri-lesional areasadjacent to the developing necrotic core demarcating the region of majordamage. RGM⁺ cells were counted in ten high power fields (HPF, ×200magnification with an eye-piece-grid representing 0.25 mm²).

[0148] Results

[0149] 21 brains of patients with focal cerebral infarctions (FCI), 25brains with traumatic brain injury (TBI) and 4 control brains wereevaluated for RGM protein expression by immunohistochemistry.

[0150] 1. Healthy, Neuropathological Unaltered Control Brains

[0151] In control brains without neuropathologically alterations, RGMimmunoreactivity was detected on white matter fibres, oliogodendrocytes,the perikarya of some neurons and RGM⁺ cells were also detected in thechoroid plexus (FIG. 8) and ependyma. Only single cells were detected inperi-vascular spaces. Further, some smooth muscle cells and fewendothelial cells but no astrocytes were labelled.

[0152] 2. Focal Cerebral Ischemia (FCI)

[0153] It was analysed whether number and distribution of RGM expressingcells is altered after cerebral infarctions. Results suggested, that RGMexpression is lesion-associated. Cellular RGM expression was confined toneurons, few reactive astrocytes and invading leukocytes. With theageing of the lesions, RGM-positive extracellular laminae componentswere found in the constituting scar.

[0154] RGM-positive cells accumulated in infarctioned white matter,hemorrhagic areas, infarction core and peri-infarctional areas,respectively. Using the students t-test, a significantly (P<0.0001)higher number of RGM⁺ cells was detected in peri-infarcional areas(MLC=24, SEM=1.1) than in remote areas (MLC=2, SEM=0.2) or controltissue (MLC=6, SEM=0.8). The morphological described peri-infarctionalareas were part of the physiologically defined penumbra. In these areasthe number of RGM-positive cells accumulated already up to day 1(p<0.0001, MLC=31.93, SEM=2.3) reached their maximum 1.5-2.5 days(MLC=34, SEM=3.2) after infarction and remained elevated up to severalweeks and months of survival (MLC=11, SEM=1.4). Early after ischemicdamage (up to 2.5 days), RGM immunoreactivity was predominantly found onneurons and leukocytes of granulocytic, monocytic and lymphocytic originin vessels within ischemic tissue. Paralleled by edema formation, up to1-7 days, RGM-positive cells were found extravasating outside thevascular walls into the focal ischemic lesioned parenchyma. Inperivascular regions, RGM-positive cells formed clusters in theVirchow-Robin spaces from day 1-7, which subsided later. Theseperi-vascular cells, also referred to as adventitial or perithelialcells are characteristically alert immune cells (Kato and Walz, 2000;Streit et al., 1999). With lesion aging, from day 3 onwards, lesionalRGM expression by few reactive astrocytes, was observed. At laterstages, arising 1 week after infarction, extracellular RGM deposits weredetected constituting neo-laminae localized to areas of ongoing scarformation. These RGM-positive laminae increased in magnitude andregional extend over time. With tissue reorganisation of the lesion,also “foamy”, lipid loaded RGM-positive phagocytic RGM-positivemicroglia/macrophages were observed.

[0155] Upregulation of cellular RGM expression correlated with the timecourse and appearance of infiltrating leukocytes and activation ofmicroglia/macrophages after injury (Stoll et al., 1998). Whereasupregulation of extracellular RGM expression correlated with the timecourse and the appearance of the scar after injury. In few cases (<5% ofcounterstained nuclei) some reactive astrocytes restricted to thedemarcating lesion core also expressed RGM.

[0156] 3. Traumatic Brain Injury

[0157] In patients who died after TBI, in accordance to cerebralinfarction (FCI) the immunohistological evaluation revealed earlycellular membranous, cytoplasmatic and nuclear RGM expression byleukocytes, few reactive astrocytes and neurons with strong staining oftheir perikarya, dendrites and axons (FIG. 9). During the observed timepost TBI, within the necrotic core and the bordering peri-necroticparenchyma accumulation of RGM-positive cells (p<0.0001) was detected inborder zones (MLC=22, SEM=0.7) compared to remote areas (MLC=1, SEM=0.1)and normal brain controls (MLC=5.8, SEM=0.8). Following TBI,RGM-positive cell numbers arose already during the first 24 hours(p<0.0001) where RGM-positive cell numbers reached maximum levels(MLC=29, SEM=0.9) and decreased subsequently. With increasing time afterTBI, most remarkable changes corresponded to areas of ongoing scarformation (FIG. 10). In these areas, well defined extracellularRGM-positive laminae were visible condensing adjacent to the borderzone. RGM immunoreactivity was also detected in endothelial and vascularsmooth muscle cells (SMC) but no significant differences were observedbetween injured and control brains.

[0158] References as mentioned in the example herein above:

[0159] Beschomer, Acta Neuropathol. 100 (2000), 377-384

[0160] Graham, “Greenfield's Neuropathology.” D. I. Graham and P. L.Lantos (eds), 6^(th). Edn., Edward Arnold, London (1996), pps. 197-248

[0161] Kalimo, Greenfield's Neuropathology 6^(th). Edn. Arnold, LondonSydney Auckland (1996), pp 315-381.

[0162] Kato, Brain Pathol., 10 (2000), 137-143.

[0163] Postler, Glia 19 (1997), 27-34.

[0164] Schwab, Acta Neuropathol. 99 (2000), 609-614.

[0165] Stoll, Prog. Neurobiol. 56 (1998), 149-171.

[0166] Streit, Prog. Neurobiol. 57 (1999), 563-581.

EXAMPLE VI Change of Tumor Growth Behaviour in Mice

[0167] Hybridoma cells secreting the RGM-specific F3D4 monoclonalantibody were injected into the peritoneum of mice, primed with mineraloil. Normally the hybridoma cells continue to divide in the peritoneumand the hybridoma cells secreted large amounts of antibody, resulting information of large ascites tumors. Mice receiving the F3D4-producinghybridoma cells did not develop ascites tumors in the peritoneal cavity,but developed solid, adherent tumors. The F3D4 monoclonal antibodyresulted in a change of phenotype of the tumorigenic hybridoma cellsfrom a less invasive, non-adherent state to an invasive adherent state.Masking of endogeneous RGM by the antibodies secreted from the hybridomacells, enabled adhesion and invasion of these tumor cells and wasresponsible for this outcome.

EXAMPLE VII Detection of a Functional RGM Fragment

[0168] RGM was cloned into the pTriEx vector and the vector was cutinside the polylinker side and inside the RGM sequence using SacI in thefirst step. After ligation of both ends the RGM containing vector wascut in the second step with Stul inside the RGM sequence and in thepolylinker with PmII. After ligation of both ends, the vector with theshorter RGM-fragments was transfected into COS7 cells. Cell lysates ofthese COS cells were purified using an anti-RGM1 affinity column andRGM-containing fractions were used in collapse assay experiments. Afragment as described in SEQ ID NO:19 was active in said assays.

EXAMPLE VIII Detection of Further RGMs

[0169] A publicly available computer database at the National Center forBiotechnology Information (NCBI, USA) was used to identify human geneshomologous to chicken RGM, employing the information and dataillustrated in the examples herein above. A search strategy based on theBlast algorithm (NCBI) resulted in three human genes located onchromosomes 1, 5 and 15. The corresponding contigs are NT_(—)021932.5(RGM3), NT_(—)029283.2 (RGM2) and NT_(—010370.5) (RGM1), respectively.cDNA sequences for RGM 1, 2 and 3 were derived from these genomicsequences by omitting introns and fusing the remaining exons.

[0170] Corresponding aminio acid and nucleotide sequences for human RGM2are illustrated in appended SEQ ID NOs: 22 and 23. Human RGM3-sequencesare shown in SEQ ID NOs: 24 and 25.

1 25 1 9 PRT Artificial Sequence Description of Artificial Sequencesynthetic 1 Tyr Leu Gly Thr Thr Leu Val Val Arg 1 5 2 7 PRT ArtificialSequence Description of Artificial Sequence synthetic 2 Thr Phe Thr AspThr Phe Gln 1 5 3 12 PRT Artificial Sequence Description of ArtificialSequence synthetic 3 Met Pro Glu Glu Val Val Asn Ala Val Glu Asp Arg 1 510 4 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic 4 Leu Thr Leu Leu Phe Lys 1 5 5 10 PRT Artificial SequenceDescription of Artificial Sequence synthetic 5 Thr Phe Thr Asp Thr PheGln Thr Cys Lys 1 5 10 6 14 PRT Artificial Sequence Description ofArtificial Sequence synthetic 6 Gly Cys Pro Leu Asn Gln Gln Leu Asp PheGln Thr Met Arg 1 5 10 7 5 PRT Artificial Sequence Description ofArtificial Sequence synthetic 7 Ala Glu Met Asp Glu 1 5 8 7 PRTArtificial Sequence Description of Artificial Sequence synthetic 8 ProGlu Ala Phe Thr Tyr Glu 1 5 9 5 PRT Artificial Sequence Description ofArtificial Sequence synthetic 9 His Leu Glu Tyr Arg 1 5 10 5 PRTArtificial Sequence Description of Artificial Sequence synthetic 10 GlnGly Leu Tyr Leu 1 5 11 29 DNA Artificial Sequence Description ofArtificial Sequence synthetic 11 atgccagctg aaggaaggta gctgtagct 29 1231 DNA Artificial Sequence Description of Artificial Sequence synthetic12 ttagctacag ctaccttcct tcagctggca t 31 13 30 DNA Artificial SequenceDescription of Artificial Sequence synthetic 13 gactacagct ttctcaagacagcttgctaa 30 14 30 DNA Artificial Sequence Description of ArtificialSequence synthetic 14 ttagcaagct gtcttgagaa agctgtagtc 30 15 29 DNAArtificial Sequence Description of Artificial Sequence synthetic 15aactcaagca agcttagctg actttctca 29 16 29 DNA Artificial SequenceDescription of Artificial Sequence synthetic 16 tgagaaagtc agctaagcttgcttgagtt 29 17 1302 DNA Artificial Sequence Description of ArtificialSequence synthetic 17 atgggtatgg ggagaggggc aggatccaca gccctgggacttttccaaat cctccctgtc 60 tttctctgca tcttccctcc agtgacgtct ccatgcaagatcctcaagtg caactctgag 120 ttctgggcgg ccacgtcggg ttcgcaccac ctgggcgcagaggaaacccc ggagttctgc 180 acggcgttgc gcgcctacgc gcactgcacc cgccgcaccgcccgcacctg caggggggac 240 ctggcctacc actcggccgt gcatggcata gacgatctcatggtgcaaca caactgctcc 300 aaggatggcc ccacgtccca gccccgcctc cggacattgccccccgggga cagccaggag 360 cgctctgaca gccccgaaat ctgccactac gagaagagctttcacaaaca ctcggcagct 420 cccaactaca cccactgtgg gctcttcggg gacccccacctcaggacttt cacggacacc 480 ttccagacct gcaaggtgca aggggcttgg ccgctcatagacaataacta cctgaacgtc 540 caggtcacca acacgccggt gctgcctggc tcctcagccaccgccaccag caagctcacc 600 atcatcttca agagcttcca ggaatgcgtg gagcagaaagtgtaccaggc agagatggac 660 gagctccctg ctgcctttgc tgatggctcc aagaacggcggcgacaagca cggagccaac 720 agcctgaaga tcaccgagaa ggtgtcgggc cagcacatcgagatccaggc caagtacatt 780 ggcaccacca tcgtggtgag gcaggtgggc cgctacctcaccttcgccgt gcgtatgccg 840 gaggaggtgg tcaacgctgt ggaggaccgg gacagtcagggcctctacct gtgcctccgg 900 ggttgtccgc tcaaccaaca gattgacttc cagactttccgcttggctca ggccgctgag 960 ggccgtgctc gcaggaaggg gcccagcttg ccggccccccctgaggcctt cacttacgag 1020 tcggccactg ccaagtgcag ggaaaagctg cccgtagaggacctctactt ccagtcctgc 1080 gtctttgacc tcctgactac gggggatgtc aacttcatgctggctgctta ttacgctttt 1140 gaggacgtga agatgcttca ctccaacaaa gacaaactgcacctctatga aaggacacgg 1200 gccctagccc cgggcaatgc agctccctcg gagcatccctgggccctccc tgccctctgg 1260 gtagcactgc tgagtttgag tcagtgttgg ttgggtttgtta 1302 18 434 PRT Gallus gallus 18 Met Gly Met Gly Arg Gly Ala Gly SerThr Ala Leu Gly Leu Phe Gln 1 5 10 15 Ile Leu Pro Val Phe Leu Cys IlePhe Pro Pro Val Thr Ser Pro Cys 20 25 30 Lys Ile Leu Lys Cys Asn Ser GluPhe Trp Ala Ala Thr Ser Gly Ser 35 40 45 His His Leu Gly Ala Glu Glu ThrPro Glu Phe Cys Thr Ala Leu Arg 50 55 60 Ala Tyr Ala His Cys Thr Arg ArgThr Ala Arg Thr Cys Arg Gly Asp 65 70 75 80 Leu Ala Tyr His Ser Ala ValHis Gly Ile Asp Asp Leu Met Val Gln 85 90 95 His Asn Cys Ser Lys Asp GlyPro Thr Ser Gln Pro Arg Leu Arg Thr 100 105 110 Leu Pro Pro Gly Asp SerGln Glu Arg Ser Asp Ser Pro Glu Ile Cys 115 120 125 His Tyr Glu Lys SerPhe His Lys His Ser Ala Ala Pro Asn Tyr Thr 130 135 140 His Cys Gly LeuPhe Gly Asp Pro His Leu Arg Thr Phe Thr Asp Thr 145 150 155 160 Phe GlnThr Cys Lys Val Gln Gly Ala Trp Pro Leu Ile Asp Asn Asn 165 170 175 TyrLeu Asn Val Gln Val Thr Asn Thr Pro Val Leu Pro Gly Ser Ser 180 185 190Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile Phe Lys Ser Phe Gln Glu 195 200205 Cys Val Glu Gln Lys Val Tyr Gln Ala Glu Met Asp Glu Leu Pro Ala 210215 220 Ala Phe Ala Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly Ala Asn225 230 235 240 Ser Leu Lys Ile Thr Glu Lys Val Ser Gly Gln His Ile GluIle Gln 245 250 255 Ala Lys Tyr Ile Gly Thr Thr Ile Val Val Arg Gln ValGly Arg Tyr 260 265 270 Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val ValAsn Ala Val Glu 275 280 285 Asp Arg Asp Ser Gln Gly Leu Tyr Leu Cys LeuArg Gly Cys Pro Leu 290 295 300 Asn Gln Gln Ile Asp Phe Gln Thr Phe ArgLeu Ala Gln Ala Ala Glu 305 310 315 320 Gly Arg Ala Arg Arg Lys Gly ProSer Leu Pro Ala Pro Pro Glu Ala 325 330 335 Phe Thr Tyr Glu Ser Ala ThrAla Lys Cys Arg Glu Lys Leu Pro Val 340 345 350 Glu Asp Leu Tyr Phe GlnSer Cys Val Phe Asp Leu Leu Thr Thr Gly 355 360 365 Asp Val Asn Phe MetLeu Ala Ala Tyr Tyr Ala Phe Glu Asp Val Lys 370 375 380 Met Leu His SerAsn Lys Asp Lys Leu His Leu Tyr Glu Arg Thr Arg 385 390 395 400 Ala LeuAla Pro Gly Asn Ala Ala Pro Ser Glu His Pro Trp Ala Leu 405 410 415 ProAla Leu Trp Val Ala Leu Leu Ser Leu Ser Gln Cys Trp Leu Gly 420 425 430Leu Leu 19 116 PRT Gallus gallus 19 Glu Leu Pro Ala Ala Phe Ala Asp GlySer Lys Asn Gly Gly Asp Lys 1 5 10 15 His Gly Ala Asn Ser Leu Lys IleThr Glu Lys Val Ser Gly Gln His 20 25 30 Ile Glu Ile Gln Ala Lys Tyr IleGly Thr Thr Ile Val Val Arg Gln 35 40 45 Val Gly Arg Tyr Leu Thr Phe AlaVal Arg Met Pro Glu Glu Val Val 50 55 60 Asn Ala Val Glu Asp Arg Asp SerGln Gly Leu Tyr Leu Cys Leu Arg 65 70 75 80 Gly Cys Pro Leu Asn Gln GlnIle Asp Phe Gln Thr Phe Arg Leu Ala 85 90 95 Gln Ala Ala Glu Gly Arg AlaArg Arg Lys Gly Pro Ser Leu Pro Ala 100 105 110 Pro Pro Glu Ala 115 20434 PRT Homo sapiens 20 Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala LeuGly Phe Trp Pro 1 5 10 15 Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro AlaAla Thr Ser Pro Cys 20 25 30 Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp SerAla Thr Ser Gly Ser 35 40 45 His Ala Pro Ala Ser Asp Asp Thr Pro Glu PheCys Ala Ala Leu Arg 50 55 60 Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala ArgThr Cys Arg Gly Asp 65 70 75 80 Leu Ala Tyr His Ser Ala Val His Gly IleGlu Asp Leu Met Ser Gln 85 90 95 His Asn Cys Ser Lys Asp Gly Pro Thr SerGln Pro Arg Leu Arg Thr 100 105 110 Leu Pro Pro Ala Gly Asp Ser Gln GluArg Ser Asp Ser Pro Glu Ile 115 120 125 Cys His Tyr Glu Lys Ser Phe HisLys His Ser Ala Thr Pro Asn Tyr 130 135 140 Thr His Cys Gly Leu Phe GlyAsp Pro His Leu Arg Thr Phe Thr Asp 145 150 155 160 Arg Phe Gln Thr CysLys Val Gln Gly Ala Trp Pro Leu Ile Asp Asn 165 170 175 Asn Tyr Leu AsnVal Gln Val Thr Asn Thr Pro Val Leu Pro Gly Ser 180 185 190 Ala Ala ThrAla Thr Ser Lys Leu Thr Ile Ile Phe Lys Asn Phe Gln 195 200 205 Glu CysVal Asp Gln Lys Val Tyr Gln Ala Glu Met Asp Glu Leu Pro 210 215 220 AlaAla Phe Val Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly Ala 225 230 235240 Asn Ser Leu Lys Ile Thr Glu Lys Val Ser Gly Gln His Val Glu Ile 245250 255 Gln Ala Lys Tyr Ile Gly Thr Thr Ile Val Val Arg Gln Val Gly Arg260 265 270 Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val Val Asn AlaVal 275 280 285 Glu Asp Trp Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg GlyCys Pro 290 295 300 Leu Asn Gln Gln Ile Asp Phe Gln Ala Phe His Thr AsnAla Glu Gly 305 310 315 320 Thr Gly Ala Arg Arg Leu Ala Ala Ala Ser ProAla Pro Thr Ala Pro 325 330 335 Glu Thr Phe Pro Tyr Glu Thr Ala Val AlaLys Cys Lys Glu Lys Leu 340 345 350 Pro Val Glu Asp Leu Tyr Tyr Gln AlaCys Val Phe Asp Leu Leu Thr 355 360 365 Thr Gly Asp Val Asn Phe Thr LeuAla Ala Tyr Tyr Ala Leu Glu Asp 370 375 380 Val Lys Met Leu His Ser AsnLys Asp Lys Leu His Leu Tyr Glu Arg 385 390 395 400 Thr Arg Asp Leu ProGly Arg Ala Ala Ala Gly Leu Pro Leu Ala Pro 405 410 415 Arg Pro Leu LeuGly Ala Leu Val Pro Leu Leu Ala Leu Leu Pro Val 420 425 430 Phe Cys 211305 DNA Homo sapiens 21 atgggtatgg ggagaggggc aggacgttca gccctgggattctggccgac cctcgccttc 60 cttctctgca gcttccccgc agccacctcc ccgtgcaagatcctcaagtg caactctgag 120 ttctggagcg ccacgtcggg cagccacgcc ccagcctcagacgacacccc cgagttctgt 180 gcagccttgc gcagctacgc cctgtgcacg cggcggacggcccgcacctg ccggggtgac 240 ctggcctacc actcggccgt ccatggcata gaggacctcatgagccagca caactgctcc 300 aaggatggcc ccacctcgca gccacgcctg cgcacgctcccaccggccgg agacagccag 360 gagcgctcgg acagccccga gatctgccat tacgagaagagctttcacaa gcactcggcc 420 acccccaact acacgcactg tggcctcttc ggggacccacacctcaggac tttcaccgac 480 cgcttccaga cctgcaaggt gcagggcgcc tggccgctcatcgacaataa ttacctgaac 540 gtgcaggtca ccaacacgcc tgtgctgccc ggctcagcggccactgccac cagcaagctc 600 accatcatct tcaagaactt ccaggagtgt gtggaccagaaggtgtacca ggctgagatg 660 gacgagctcc cggccgcctt cgtggatggc tctaagaacggtggggacaa gcacggggcc 720 aacagcctga agatcactga gaaggtgtca ggccagcacgtggagatcca ggccaagtac 780 atcggcacca ccatcgtggt gcgccaggtg ggccgctacctgacctttgc cgtccgcatg 840 ccagaggaag tggtcaatgc tgtggaggac tgggacagccagggtctcta cctctgcctg 900 cggggctgcc ccctcaacca gcagatcgac ttccaggccttccacaccaa tgctgagggc 960 accggtgccc gcaggctggc agccgccagc cctgcacccacagcccccga gaccttccca 1020 tacgagacag ccgtggccaa gtgcaaggag aagctgccggtggaggacct gtactaccag 1080 gcctgcgtct tcgacctcct caccacgggc gacgtgaacttcacactggc cgcctactac 1140 gcgttggagg atgtcaagat gctccactcc aacaaagacaaactgcacct gtatgagagg 1200 actcgggacc tgccaggcag ggcggctgcg gggctgcccctggccccccg gcccctcctg 1260 ggcgccctcg tcccgctcct ggccctgctc cctgtgttctgctag 1305 22 1314 DNA Homo sapiens 22 atgggcttga gagcagcacc ttccagcgccgccgctgccg ccgccgaggt tgagcagcgc 60 cgcagccccg ggctctgccc cccgccgctggagctgctgc tgctgctgct gttcagcctc 120 gggctgctcc acgcaggtga ctgccaacagccagcccaat gtcgaatcca gaaatgcacc 180 acggacttcg tgtccctgac ttctcacctgaactctgccg ttgacggctt tgactctgag 240 ttttgcaagg ccttgcgtgc ctatgctggctgcacccagc gaacttcaaa agcctgccgt 300 ggcaacctgg tataccattc tgccgtgttgggtatcagtg acctcatgag ccagaggaat 360 tgttccaagg atggacccac atcctctaccaaccccgaag tgacccatga tccttgcaac 420 tatcacagcc acgctggagc cagggaacacaggagagggg accagaaccc tcccagttac 480 cttttttgtg gcttgtttgg agatcctcacctcagaactt tcaaggataa cttccaaaca 540 tgcaaagtag aaggggcctg gccactcatagataataatt atctttcagt tcaagtgaca 600 aacgtacctg tggtccctgg atccagtgctactgctacaa ataagatcac tattatcttc 660 aaagcccacc atgagtgtac agatcagaaagtctaccaag ctgtgacaga tgacctgccg 720 gccgcctttg tggatggcac caccagtggtggggacagcg atgccaagag cctgcgtatc 780 gtggaaaggg agagtggcca ctatgtggagatgcacgccc gctatatagg gaccacagtg 840 tttgtgcggc aggtgggtcg ctacctgacccttgccatcc gtatgcctga agacctggcc 900 atgtcctacg aggagagcca ggacctgcagctgtgcgtga acggctgccc cctgagtgaa 960 cgcatcgatg acgggcaggg ccaggtgtctgccatcctgg gacacagcct gcctcgcacc 1020 tccttggtgc aggcctggcc tggctacacactggagactg ccaacactca atgccatgag 1080 aagatgccag tgaaggacat ctatttccagtcctgtgtct tcgacctgct caccactggt 1140 gatgccaact ttactgccgc agcccacagtgccttggagg atgtggaggc cctgcaccca 1200 aggaaggaac gctggcacat tttccccagcagtggcaatg ggactccccg tggaggcagt 1260 gatttgtctg tcagtctagg actcacctgcttgatcctta tcgtgttttt gtag 1314 23 437 PRT Homo sapiens 23 Met Gly LeuArg Ala Ala Pro Ser Ser Ala Ala Ala Ala Ala Ala Glu 1 5 10 15 Val GluGln Arg Arg Ser Pro Gly Leu Cys Pro Pro Pro Leu Glu Leu 20 25 30 Leu LeuLeu Leu Leu Phe Ser Leu Gly Leu Leu His Ala Gly Asp Cys 35 40 45 Gln GlnPro Ala Gln Cys Arg Ile Gln Lys Cys Thr Thr Asp Phe Val 50 55 60 Ser LeuThr Ser His Leu Asn Ser Ala Val Asp Gly Phe Asp Ser Glu 65 70 75 80 PheCys Lys Ala Leu Arg Ala Tyr Ala Gly Cys Thr Gln Arg Thr Ser 85 90 95 LysAla Cys Arg Gly Asn Leu Val Tyr His Ser Ala Val Leu Gly Ile 100 105 110Ser Asp Leu Met Ser Gln Arg Asn Cys Ser Lys Asp Gly Pro Thr Ser 115 120125 Ser Thr Asn Pro Glu Val Thr His Asp Pro Cys Asn Tyr His Ser His 130135 140 Ala Gly Ala Arg Glu His Arg Arg Gly Asp Gln Asn Pro Pro Ser Tyr145 150 155 160 Leu Phe Cys Gly Leu Phe Gly Asp Pro His Leu Arg Thr PheLys Asp 165 170 175 Asn Phe Gln Thr Cys Lys Val Glu Gly Ala Trp Pro LeuIle Asp Asn 180 185 190 Asn Tyr Leu Ser Val Gln Val Thr Asn Val Pro ValVal Pro Gly Ser 195 200 205 Ser Ala Thr Ala Thr Asn Lys Ile Thr Ile IlePhe Lys Ala His His 210 215 220 Glu Cys Thr Asp Gln Lys Val Tyr Gln AlaVal Thr Asp Asp Leu Pro 225 230 235 240 Ala Ala Phe Val Asp Gly Thr ThrSer Gly Gly Asp Ser Asp Ala Lys 245 250 255 Ser Leu Arg Ile Val Glu ArgGlu Ser Gly His Tyr Val Glu Met His 260 265 270 Ala Arg Tyr Ile Gly ThrThr Val Phe Val Arg Gln Val Gly Arg Tyr 275 280 285 Leu Thr Leu Ala IleArg Met Pro Glu Asp Leu Ala Met Ser Tyr Glu 290 295 300 Glu Ser Gln AspLeu Gln Leu Cys Val Asn Gly Cys Pro Leu Ser Glu 305 310 315 320 Arg IleAsp Asp Gly Gln Gly Gln Val Ser Ala Ile Leu Gly His Ser 325 330 335 LeuPro Arg Thr Ser Leu Val Gln Ala Trp Pro Gly Tyr Thr Leu Glu 340 345 350Thr Ala Asn Thr Gln Cys His Glu Lys Met Pro Val Lys Asp Ile Tyr 355 360365 Phe Gln Ser Cys Val Phe Asp Leu Leu Thr Thr Gly Asp Ala Asn Phe 370375 380 Thr Ala Ala Ala His Ser Ala Leu Glu Asp Val Glu Ala Leu His Pro385 390 395 400 Arg Lys Glu Arg Trp His Ile Phe Pro Ser Ser Gly Asn GlyThr Pro 405 410 415 Arg Gly Gly Ser Asp Leu Ser Val Ser Leu Gly Leu ThrCys Leu Ile 420 425 430 Leu Ile Val Phe Leu 435 24 1272 DNA Homo sapiens24 atgggccagt cccctagtcc caggtcctcc catggcagtc ccccaactct aagcactctc 60actctcctgc tgctcctctg tggacatgct cattctcaat gcaagatcct ccgctgcaat 120gctgagtacg tatcgtccac tctgagcctt agaggtgggg gttcatcagg agcacttcga 180ggaggaggag gaggaggccg gggtggaggg gtgggctctg gcggcctctg tcgagccctc 240cgctcctatg cgctctgcac tcggcgcacc gcccgcacct gccgcgggga cctcgccttc 300cattcggcgg tacatggcat cgaagacctg atgatccagc acaactgctc ccgccagggc 360cctacagccc ctcccccgcc ccggggcccc gcccttccag gcgcgggctc cggcctccct 420gccccggacc cttgtgacta tgaaggccgg ttttcccggc tgcatggtcg tcccccgggg 480ttcttgcatt gcgcttcctt cggggacccc catgtgcgca gcttccacca tcactttcac 540acatgccgtg tccaaggagc ttggcctcta ctggataatg acttcctctt tgtccaagcc 600accagctccc ccatggcgtt gggggccaac gctaccgcca cccggaagct caccatcata 660tttaagaaca tgcaggaatg cattgatcag aaggtgtatc aggctgaggt ggataatctt 720cctgtagcct ttgaagatgg ttctatcaat ggaggtgacc gacctggggg atccagtttg 780tcgattcaaa ctgctaaccc tgggaaccat gtggagatcc aagctgccta cattggcaca 840actataatca ttcggcagac agctgggcag ctctccttct ccatcaaggt agcagaggat 900gtggccatgg ccttctcagc tgaacaggac ctgcagctct gtgttggggg gtgccctcca 960agtcagcgac tctctcgatc agagcgcaat cgtcggggag ctataaccat tgatactgcc 1020agacggctgt gcaaggaagg gcttccagtg gaagatgctt acttccattc ctgtgtcttt 1080gatgttttaa tttctggtga tcccaacttt accgtggcag ctcaggcagc actggaggat 1140gcccgagcct tcctgccaga cttagagaag ctgcatctct tcccctcaga tgctggggtt 1200cctctttcct cagcaaccct cttagctcca ctcctttctg ggctctttgt tctgtggctt 1260tgcattcagt aa 1272 25 423 PRT Homo sapiens 25 Met Gly Gln Ser Pro SerPro Arg Ser Ser His Gly Ser Pro Pro Thr 1 5 10 15 Leu Ser Thr Leu ThrLeu Leu Leu Leu Leu Cys Gly His Ala His Ser 20 25 30 Gln Cys Lys Ile LeuArg Cys Asn Ala Glu Tyr Val Ser Ser Thr Leu 35 40 45 Ser Leu Arg Gly GlyGly Ser Ser Gly Ala Leu Arg Gly Gly Gly Gly 50 55 60 Gly Gly Arg Gly GlyGly Val Gly Ser Gly Gly Leu Cys Arg Ala Leu 65 70 75 80 Arg Ser Tyr AlaLeu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly 85 90 95 Asp Leu Ala PheHis Ser Ala Val His Gly Ile Glu Asp Leu Met Ile 100 105 110 Gln His AsnCys Ser Arg Gln Gly Pro Thr Ala Pro Pro Pro Pro Arg 115 120 125 Gly ProAla Leu Pro Gly Ala Gly Ser Gly Leu Pro Ala Pro Asp Pro 130 135 140 CysAsp Tyr Glu Gly Arg Phe Ser Arg Leu His Gly Arg Pro Pro Gly 145 150 155160 Phe Leu His Cys Ala Ser Phe Gly Asp Pro His Val Arg Ser Phe His 165170 175 His His Phe His Thr Cys Arg Val Gln Gly Ala Trp Pro Leu Leu Asp180 185 190 Asn Asp Phe Leu Phe Val Gln Ala Thr Ser Ser Pro Met Ala LeuGly 195 200 205 Ala Asn Ala Thr Ala Thr Arg Lys Leu Thr Ile Ile Phe LysAsn Met 210 215 220 Gln Glu Cys Ile Asp Gln Lys Val Tyr Gln Ala Glu ValAsp Asn Leu 225 230 235 240 Pro Val Ala Phe Glu Asp Gly Ser Ile Asn GlyGly Asp Arg Pro Gly 245 250 255 Gly Ser Ser Leu Ser Ile Gln Thr Ala AsnPro Gly Asn His Val Glu 260 265 270 Ile Gln Ala Ala Tyr Ile Gly Thr ThrIle Ile Ile Arg Gln Thr Ala 275 280 285 Gly Gln Leu Ser Phe Ser Ile LysVal Ala Glu Asp Val Ala Met Ala 290 295 300 Phe Ser Ala Glu Gln Asp LeuGln Leu Cys Val Gly Gly Cys Pro Pro 305 310 315 320 Ser Gln Arg Leu SerArg Ser Glu Arg Asn Arg Arg Gly Ala Ile Thr 325 330 335 Ile Asp Thr AlaArg Arg Leu Cys Lys Glu Gly Leu Pro Val Glu Asp 340 345 350 Ala Tyr PheHis Ser Cys Val Phe Asp Val Leu Ile Ser Gly Asp Pro 355 360 365 Asn PheThr Val Ala Ala Gln Ala Ala Leu Glu Asp Ala Arg Ala Phe 370 375 380 LeuPro Asp Leu Glu Lys Leu His Leu Phe Pro Ser Asp Ala Gly Val 385 390 395400 Pro Leu Ser Ser Ala Thr Leu Leu Ala Pro Leu Leu Ser Gly Leu Phe 405410 415 Val Leu Trp Leu Cys Ile Gln 420

1. Use of a modulator of a polypeptide having or comprising the aminoacid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragmentor derivative thereof or of a polynucleotide encoding said polypeptideor fragment or derivative for the preparation of a pharmaceuticalcomposition for preventing, alleviating or treating diseases orconditions associated with the degeneration or injury of vertebratenervous tissue, associated with seizures associated with angiogenicdisorders or disorders of the cardio-vascular system.
 2. The use ofclaim 1 wherein said diseases or conditions associated with thedegeneration or injury of vertebrate nervous tissue are selected fromthe group consisting of neurodegenerative diseases, nerve fiber injuriesand disorders related to nerve fiber losses.
 3. The use of claim 2,wherin said neurodegenerative disease is selected from the groupconsisting of motorneuronal diseases (MND), ALS, Alzheimers disease,Parkinsons disease, progressive bulbar palsy, progressive muscularatrophy, HIV-related dementia and spinal muscular atrophy(ies), Down'sSyndrome, Huntington's Disease, Creutzfeldt-Jacob Disease,Gerstmann-Straeussler Syndrome, kuru, Scrapie, transmissible minkencephalopathy, other unknown prion diseases, multiple system atrophy,Riley-Day familial dysautonomia wherein said nerve fiber injuries areselected from the group consisting of spinal cord injury(ies), braininjuries related to raised intracranial pressure, trauma, secondarydamage due to increased intracranial pressure, infection, infarction,exposure to toxic agents, malignancy and paraneoplastic syndromes andwherein said disorders related to nerve fiber losses are selected fromthe group consisting of paresis of nervus facialis, nervus medianus,nervus ulnaris, nervus axillaris, nervus thoracicus longus, nervusradialis and for of other peripheral nerves.
 4. The use of claim 1,wherein said disease or condition associated with seizures is epilepsy.5. The use of claim 1, wherein said disease or conditions associatedwith angiogenic disorders are selected from the group consisting ofischemic disorders, infarction, disorders leading to the progression ofvascular plaque formation in cardiovascular, cerebrovascular and/ornephro-vascular disorders or disorders wherein the permeability of theblood-brain barrier has to be regulated.
 6. The use of claim 1, whereinsaid disorders of the cardiovascular system comprises disorders of theblood-brain barrier, brain oedema, secondary brain damages due toincreased intracranial pressure, infection, infarction, ischemia,hypoxia, hypoglycemia, exposure to toxic agents, malignancy,paraneoplastic syndromes.
 7. Use of a modulator of a polypeptide havingor comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 orof a functional fragment or derivative thereof or of a polynucleotideencoding said polypeptide or fragment or derivative for the preparationof a pharmaceutical composition for remyelinization.
 8. Use of amodulator of a polypeptide having or comprising the amino acid sequenceof SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivativethereof or of a polynucleotide encoding said polypeptide or fragment orderivative for modifying and/or altering the differentiation of neuronalstem cells and/or their progenitors.
 9. The use of any one of claims 1to 8, wherein said modulator is an antibody or a fragment or aderivative thereof, is an aptamer, is a specific receptor moleculecapable of interacting with a polypeptide having or comprising the aminoacid sequence of SEQ ID NO: 18, 20, 23 or 25 or with a functionalfragment or derivative thereof or is a specific nucleic acid moleculeinteracting with a polynucleotide encoding said polypeptide.
 10. Use ofa polypeptide having or comprising the amino acid sequence of SEQ ID NOs18, 20, 23 or 25 or of a functional fragment or derivative thereof or ofa polynucleotide encoding such polypeptide or fragment as derivative forthe preparation of a pharmaceutical composition for preventing ortreating diseases or conditions associated with excessive collateralsprouting of nerve fibres.
 11. Use of a polypeptide having or comprisingthe amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of afunctional fragment or derivative thereof or of a polynucleotideencoding said polypeptide or fragment or derivative for the preparationof a pharmaceutical composition for preventing or treating tumor growthor formation of tumor metastases.
 12. Use of a polypeptide having orcomprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or ofa functional fragment or derivative thereof or of a polynucleotideencoding said polypeptide or fragment or derivative for the preparationof a pharmaceutical composition for preventing, alleviating or treatingdiseases or conditions associated with the activity of autoreactiveimmune cells or with overreactive inflammatory cells.
 13. Use of apolypeptide having or comprising the amino acid sequence of SEQ ID NOs18, 20, 23 or 25 or of a functional fragment or derivative thereof or ofa polynucleotide encoding said polypeptide or fragment or derivative forthe preparation of a pharmaceutical composition for the treatment ofinflammation processes and/or allergies, for wound healing or for thesuppression/alleviation of scar formation.
 14. Use of a polypeptidehaving or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or25 or of a functional fragment or derivative thereof or of apolynucleotide encoding said polypeptide or fragment or derivative as amarker of stem cells.
 15. Use of a polypeptide having or comprising theamino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functionalfragment or derivative thereof or of a polynucleotide encoding saidpolypeptide or fragment or derivative for the preparation of apharmaceutical composition for alleviating, preventing and/or treatinghomoestatic and/or bleeding disorders and/or vascular damage.
 16. Theuse of any one of claims 10 to 15, wherein said polypeptide or fragmentor derivaitve is soluble.
 17. Use of an antibody or a fragment or aderivative thereof, is an aptamer, is a specific receptor moleculecapable of interacting with a polypeptide having or comprising the aminoacid sequence of SEQ ID NO: 18, 20, 23 or 25 or with a functionalfragment or derivative thereof, or is a specific nucleic acid moleculeinteracting with a polynucleotide encoding said polypeptide for thepreparation of a diagnostic composition for detecting neurological,neurodegenerative disorders or dispositions thereto.